Technical field
[0001] The present invention relates to the microbiological industry, and specifically to
a method for producing an L-amino acid such as L-threonine, L-lysine, L-histidine,
L-phenylalanine, L-arginine, L-tryptophan, L-glutamic acid and L-leucine by fermentation
using a bacterium with an enhanced activity of alcohol dehydrogenase.
Background art
[0002] Conventionally, L-amino acids are industrially produced by fermentation methods utilizing
strains of microorganisms obtained from natural sources, or mutants thereof. Typically,
the microorganisms are modified to enhance production yields of L-amino acids.
[0003] Many techniques to enhance L-amino acid production yields have been reported, including
transformation of microorganisms with recombinant DNA (
U.S. Patent No. 4,278,765). Other techniques for enhancing production yields include increasing the activities
of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes
to feedback inhibition by the resulting L-amino acid (
U.S. Patent Nos. 4,346,170,
5,661,012, and
6,040,160).
[0004] By optimizing the main biosynthetic pathway of a desired compound, further improvement
of L-amino acid producing strains can be accomplished. Typically, this is accomplished
via supplementation of the bacterium with increasing amounts of a carbon source such
as sugars, for example, glucose. Despite the efficiency of glucose transport by PTS,
access to the carbon source in a highly productive strain still may be insufficient.
Another way to increase productivity of L-amino acid producing strains and decrease
the cost of the target L-amino acid is to use an alternative source of carbon, such
as alcohol, for example, ethanol.
[0005] Alcohol dehydrogenase (ethanol oxidoreductase, AdhE) of
Escherichia coli is a multifunctional enzyme that catalyzes fermentative production of ethanol by
two sequential NADH-dependent reductions of acetyl-CoA, as well as deactivation of
pyruvate formate-lyase, which cleaves pyruvate to acetyl-CoA and formate.
[0006] AdhE is abundantly synthesized (about 3x10
4 copies per cell) during anaerobic growth in the presence of glucose and forms helical
structures, called spirosomes, which are around 0.22 µm long and contain 40-60 AdhE
molecules (
Kessler, D., Herth, W., and Knappe, J., J. Biol. Chem., 267, 18073-18079 (1992)). When the
E. coli cell culture is shifted from anaerobic to aerobic conditions, transcription of the
adhE gene is reduced and maintained within 10% of the range found under anaerobiosis (
Chen, Y. M., and Lin, E. C. C., J. Bacteriol. 173, 8009-8013 (1991);
Leonardo, M. R., Cunningham, P. R., and Clark, D. P., J. Bacteriol. 175, 870-878 (1993);
Mikulskis, A., Aristarkhov, A., and Lin, E. C. C., J. Bacteriol. 179, 7129-7134 (1997);
Membrillo-Hernandez, J., and Lin, E. C. C., J. Bacteriol. 181, 7571-7579 (1999)). Translation is also regulated and requires RNase III (
Membrillo-Hernandez, J., and Lin, E. C. C., J. Bacteriol. 181, 7571-7579 (1999);
Aristarkhov, A. et al, J. Bacteriol. 178,4327-4332 (1996)). AdhE has been identified as one of the major targets when E.
coli cells are subjected to hydrogen peroxide stress (
Tamarit, J., Cabiscol, E., and Ros, J., J. Biol. Chem. 273, 3027-3032 (1998)).
[0007] Despite the reversibility of the two NADH-coupled reactions catalyzed by AdhE, wild-type
E. coli is unable to grow in the presence of ethanol as the sole source of carbon and energy,
because the
adhE gene is transcribed aerobically at lowered levels (
Chen, Y. M. and Lin, E. C. C., J. Bacteriol. 73, 8009-8013 (1991);
Leonardo, M. R., Cunningham, P. R. & Clark, D. P., J. Bacteriol. 175 870-878 (1993)) and the half-life of the AdhE protein is shortened during aerobic metabolism by
metal-catalyzed oxidation (MCO).
[0008] Mutants of
E. coli capable of aerobic growth on ethanol as the sole carbon and energy source have been
isolated and characterized (mutants with the substitution Ala267Thr grew in the presence
of ethanol with a doubling time of 240 min; with the substitutions Ala267Thr and Glu568Lys,
a doubling time of 90 min at 37°C) (
Membrillo-Hernandez, J. et al, J. Biol. Chem. 275, 33869-33875 (2000);
Holland-Staley, C. A. et al, J. Bacteriol. 182, 6049-6054 (2000)). Apparently, when the two sequential reactions are catalyzed in a direction opposite
to that of the physiological one, acetyl-CoA formation is rate-limiting for wild-type
AdhE. The tradeoff for improving the V
max by the A267T substitution in AdhE is decreased thermal enzyme stability and increased
sensitivity to MCO damage. The second amino acid substitution, E568K, in AdhE (A267T/E568K)
partially restored protein stability and resistance to MCO damage without further
improvement of catalytic efficiency in substrate oxidation.
[0009] However, there have been no reports to date of using a bacterium of the
Enterobacteriaceae family which has an enhanced activity of either native alcohol dehydrogenase or mutant
alcohol dehydrogenase resistant to aerobic inactivation for increasing the production
of L-amino acids by fermentation in a culture medium containing ethanol.
SUMMARY OF THE INVENTION
[0010] Objects of the present invention include enhancing the productivity of L-amino acid-producing
strains and providing a method for producing non-aromatic or aromatic L-amino acids
using these strains.
[0011] This aim was achieved by finding that expressing either the native or mutant
adhE gene which encodes alcohol dehydrogenase under the control of a promoter which functions
under an aerobic cultivation condition enhances production of L-amino acids, for example,
L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-glutamic
acid, and/or L-leucine.
[0012] It is an object of the present invention to provide a method for producing an L-amino
acid comprising:
- A) cultivating in a culture medium containing ethanol an L-amino acid-producing bacterium
of the Enterobacteriaceae family having an alcohol dehydrogenase, and
- B) isolating the L-amino acid from the culture medium,
wherein the gene encoding said alcohol dehydrogenase is expressed under the control
of a non-native promoter which functions under aerobic cultivation conditions.
[0013] It is a further object of the present invention to provide the method described above,
wherein said non-native promoter is selected from the group consisting of P
tac, P
lac, P
trp, P
trc, P
R, and P
L.
[0014] It is a further object of the present invention to provide the method described above,
wherein said alcohol dehydrogenase is resistant to aerobic inactivation.
[0015] It is a further object of the present invention to provide the method described above,
wherein said alcohol dehydrogenase originates from a bacterium selected from the group
consisting of
Escherichia coli, Erwinia carotovora, Salmonella typhimurium, Shigella flexneri, Yersinia
pestis, Pantoea ananatis, Lactobacillus plantarum, and
Lactococcus lactis.
[0016] It is a further object of the present invention to provide the method described above,
wherein said alcohol dehydrogenase comprises the amino acid sequence set forth in
SEQ ID NO: 2, except the glutamic acid residue at position 568 is replaced with another
amino acid residue other than an aspartic acid residue.
[0017] It is a further object of the present invention to provide the method described above,
wherein said alcohol dehydrogenase comprises the amino acid sequence set forth in
SEQ ID NO: 2, except the glutamic acid residue at position 568 is replaced with a
lysine residue.
[0018] It is a further object of the present invention to provide the method described above,
wherein said alcohol dehydrogenase has at least one additional mutation which is able
to improve the growth of said bacterium in a liquid medium which contains ethanol
as the sole carbon source.
[0019] It is a further object of the present invention to provide the method described above,
wherein said additional mutation is selected from the group consisting of:
- A) replacement of the glutamic acid residue at position 560 in SEQ ID NO: 2 with another
amino acid residue;
- B) replacement of the phenylalanine residue at position 566 in SEQ ID NO: 2 with another
amino acid residue;
- C) replacement of the glutamic acid residue, the methionine residue, the tyrosine
residue, the isoleucine residue, and the alanine residue at positions 22, 236, 461,
554, and 786, respectively, in SEQ ID NO: 2 with other amino acid residues; and
- D) combinations thereof.
[0020] It is a further object of the present invention to provide the method described above,
wherein said additional mutation is selected from the group consisting of:
- A) replacement of the glutamic acid residue at position 560 in SEQ ID NO: 2 with a
lysine residue;
- B) replacement of the phenylalanine residue at position 566 in SEQ ID NO: 2 with a
valine residue;
- C) replacement of the glutamic acid residue, the methionine residue, the tyrosine
residue, the isoleucine residue, and the alanine residue at positions 22, 236, 461,
554, and 786 in SEQ ID NO: 2 with a glycine residue, a valine residue, a cysteine
residue, a serine residue, and a valine residue, respectively; and
- D) combinations thereof.
[0021] It is a further obj ect of the present invention to provide the method described
above, wherein said bacterium belongs to the genus selected from the group consisting
of
Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Providencia, Salmonella,
Serratia, Shigella, and
Morganella.
[0022] It is a further object of the present invention to provide the method described above,
wherein said L-amino acid is selected from a group consisting of L-threonine, L-lysine,
L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-glutamic acid, and L-leucine.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] Alcohol dehydrogenase is a Fe
2+-dependent multifunctional protein with an acetaldehyde-CoA dehydrogenase activity
at the N-terminal, an iron-dependent alcohol dehydrogenase activity at the C-terminal,
and a pyruvate-formate lyase deactivase activity. Synonyms include B1241, AdhC, and
Ana. Under aerobic conditions, the half-life of the active AdhE protein is shortened
during aerobic metabolism by metal-catalyzed oxidation.
[0024] In the present invention, the phrase "activity of alcohol dehydrogenase" means an
activity of catalyzing the reaction ofNAD-dependant oxidation of alcohols into aldehydes
or ketones. Alcohol dehydrogenase (EC 1.1.1.1) works well with ethanol, n-propanol,
and n-butanol. Activity of alcohol dehydrogenase can be detected and measured by,
for example, the method described by
Membrillo-Hernandez, J. et al (J. Biol. Chem. 275, 33869-33875 (2000)).
[0025] Alcohol dehydrogenase is encoded by the
adhE gene, and any
adhE gene derived from or native to bacteria belonging to the genus
Escherichia, Erwinia, Klebsiella, Salmonella, Shigella, Yershinia, Pantoea, Lactobacillus, and
Lactococcus may be used as the alcohol dehydrogenase gene in the present invention. Specific
examples of the source of the
adhE gene include bacterial strains such as
Escherichia coli, Erwinia carotovora, Salmonella enterica, Salmonella typhimurium,
Shigella flexneri, Yersinia pseudotuberculosis, Pantoea ananatis, Lactobacillus plantarum and
Lactococcus lactis. The wild-type
adhE gene which encodes alcohol dehydrogenase from
Escherichia coli has been elucidated (nucleotide numbers complementary to numbers 1294669 to 1297344
in the sequence of GenBank accession NC_000913.2, gi: 49175990). The
adhE gene is located between the
ychG and ychE ORFs on the chromosome of
E. coli K-12. Other
adhE genes which encode alcohol dehydrogenases have also been elucidated:
adhE gene from
Erwinia carotovora (nucleotide numbers 2634501 to 2637176 in the sequence of GenBank accession NC_004547.2;
gi: 50121254);
adhE gene from
Salmonella enterica (nucleotide numbers 1718612 to 1721290 in the sequence of GenBank accession NC_004631.1;
gi: 29142095);
adhE gene from
Salmonella typhimurium (nucleotide numbers 1 to 2637 in the sequence of GenBank accession U68173.1; gi:
1519723);
adhE gene from
Shigella flexneri (nucleotide numbers complement to numbers 1290816 to 1293491in the sequence of GenBank
accession NC_004741.1, gi: 30062760);
adhE gene from
Yersinia pseudotuberculosis (nucleotide numbers complement to numbers 2478099 to 2480774 in the sequence of GenBank
accession NC_006155.1; gi: 51596429),
adhE gene from
Pantoea ananatis (SEQ ID NO: 29),
adhE gene from
Lactobaccillus plantarum (UniProtKB Entry: Q88RY9_LACPL),
adhE gene from
Lactococcus lactis MG1363 (EMBL accession no. AJ001007), and the like (See Figure 2). The nucleotide
sequence of the
adhE gene from
Escherichia coli is represented by SEQ ID NO: 1. The amino acid sequence encoded by this
adhE gene is represented by SEQ ID NO: 2.
[0026] Therefore, the
adhE gene can be obtained by PCR (polymerase chain reaction; refer to
White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the known nucleotide sequence of the gene from
the
E. coli chromosome. Genes coding for alcohol dehydrogenase from other microorganisms can
be obtained in a similar manner.
[0027] The
adhE gene derived from
Escherichia coli is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 2; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 2, which has
an activity of alcohol dehydrogenase.
[0028] The
adhE gene derived from
Pantoea ananatis is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 30; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 30, which has
an activity of alcohol dehydrogenase.
[0029] The
adhE gene derived from
Shigella flexneri is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 53; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 53, which has
an activity of alcohol dehydrogenase.
[0030] The
adhE gene derived from
Yersinia pestis is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 54; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 54, which has
an activity of alcohol dehydrogenase.
[0031] The
adhE gene derived from
Erwinia carotovora is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 55; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 55, which has
an activity of alcohol dehydrogenase.
[0032] The
adhE gene derived from
Salmonella typhimurium is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 56; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 56, which has
an activity of alcohol dehydrogenase.
[0033] The
adhE gene derived from
Lactobacillus plantarum is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 57; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 57, which has
an activity of alcohol dehydrogenase.
[0034] The
adhE gene derived from
Lactococcus lactis is exemplified by a DNA which encodes the following protein (A) or (B):
- (A) a protein which has the amino acid sequence shown in SEQ ID NO: 58; or
- (B) a variant protein of the amino acid sequence shown in SEQ ID NO: 58, which has
an activity of alcohol dehydrogenase.
[0035] The phrase "variant protein" as used in the present invention means a protein which
has changes in the sequence, whether they are deletions, insertions, additions, or
substitutions of amino acids, but still maintains alcohol dehydrogenase activity at
a useful level. The number of changes in the variant protein depends on the position
in the three dimensional structure of the protein or the type of amino acid residue.
The number of changes may be 1 to 30, preferably 1 to 15, and more preferably 1 to
5, relative to the protein (A). These changes in the variants are conservative mutations
that preserve the function of the protein. In other words, these changes can occur
in regions of the protein which are not critical for the function of the protein.
This is because some amino acids have high homology to one another so the three dimensional
structure or activity is not affected by such a change. Therefore, the protein variant
(B) may be one which has an identity of not less than 70 %, preferably not less than
80 %, and more preferably not less than 90 %, and most preferably not less than 95
% with respect to the entire amino acid sequence of alcohol dehydrogenase shown in
SEQ ID NO. 2, as long as the activity of the alcohol dehydrogenase is maintained.
[0036] Homology between two amino acid sequences can be determined using the well-known
methods, for example, the computer program BLAST 2.0, which calculates three parameters:
score, identity, and similarity.
[0037] The substitution, deletion, insertion, or addition of one or several amino acid residues
should be conservative mutation(s) so that the activity is maintained. The representative
conservative mutation is a conservative substitution. Examples of conservative substitutions
include substitution of Ser or Thr for Ala, substitution of Gln, His or Lys for Arg,
substitution of Glu, Gln, Lys, His or Asp for Asn, substitution of Asn, Glu or Gln
for Asp, substitution of Ser or Ala for Cys, substitution of Asn, Glu, Lys, His, Asp
or Arg for Gln, substitution of Asn, Gln, Lys or Asp for Glu, substitution of Pro
for Gly, substitution of Asn, Lys, Gln, Arg or Tyr for His, substitution of Leu, Met,
Val or Phe for Ile, substitution of Ile, Met, Val or Phe for Leu, substitution of
Asn, Glu, Gln, His or Arg for Lys, substitution of Ile, Leu, Val or Phe for Met, substitution
of Trp, Tyr, Met, Ile or Leu for Phe, substitution of Thr or Ala for Ser, substitution
of Ser or Ala for Thr, substitution of Phe or Tyr for Trp, substitution of His, Phe
or Trp for Tyr, and substitution of Met, Ile or Leu for Val.
[0038] Data comparing the primary sequences of alcohol dehydrogenase from
Escherichia coli, Shigella flexneri, Pantoea ananatis, Yersinia pestis, Erwinia carotovora,
Salmonella typhimurium (Gram negative bacteria), and
Lactobacillus plantarum, Lactococcus lactis (Gram positive bacteria) show a high level of homology among these proteins (see
Figure 2). From this point of view, substitutions or deletions of the amino acid residues
which are identical (marked by asterisk) in all the above-mentioned proteins could
be crucial for their function. It is possible to replace similar (marked by colon)
amino acids residues by the similar amino acid residues without deterioration of the
protein activity. But modifications of other non-conserved amino acid residues may
not lead to alteration of the activity of alcohol dehydrogenase.
[0039] The DNA which encodes substantially the same protein as the alcohol dehydrogenase
described above may be obtained, for example, by modifying the nucleotide sequence
of DNA encoding alcohol dehydrogenase (SEQ ID NO: 1), for example, by means of site-directed
mutagenesis so that the nucleotide sequence responsible for one or more amino acid
residues at a specified site is deleted, substituted, inserted, or added. DNA modified
as described above may be obtained by conventionally known mutation treatments. Such
treatments include hydroxylamine treatment of the DNA encoding proteins of present
invention, or treatment of the bacterium containing the DNA with UV irradiation or
a reagent such as N-methyl-N'-nitro-N-nitrosoguanidine or nitrous acid.
[0040] A DNA encoding substantially the same protein as alcohol dehydrogenase can be obtained
by expressing DNA having a mutation as described above in an appropriate cell, and
investigating the activity of any expressed product. A DNA encoding substantially
the same protein as alcohol dehydrogenase can also be obtained by isolating a DNA
that is able to hybridize with a probe having a nucleotide sequence which contains,
for example, the nucleotide sequence shown as SEQ ID NO: 1, under stringent conditions,
and encodes a protein having alcohol dehydrogenase activity. The "stringent conditions"
referred to herein are conditions under which so-called specific hybrids are formed,
and non-specific hybrids are not formed. For example, stringent conditions can be
exemplified by conditions under which DNAs having high homology, for example, DNAs
having identity of not less than 50%, preferably not less than 60%, more preferably
not less than 70%, still more preferably not less than 80%, further preferably not
less than 90%, most preferably not less than 95%, are able to hybridize with each
other, but DNAs having identity lower than the above are not able to hybridize with
each other. Alternatively, stringent conditions may be exemplified by conditions under
which DNA is able to hybridize at a salt concentration equivalent to ordinary washing
conditions in Southern hybridization, i.e., 1 x SSC, 0.1% SDS, preferably 0.1 x SSC,
0.1% SDS, at 60°C. Duration of washing depends on the type of membrane used for blotting
and, as a rule, what is recommended by the manufacturer. For example, recommended
duration of washing for the Hybond™ N+ nylon membrane (Amersham) under stringent conditions
is 15 minutes. Preferably, washing may be performed 2 to 3 times.
[0041] A partial sequence of the nucleotide sequence of SEQ ID NO: 1 can also be used as
a probe. Probes may be prepared by PCR using primers based on the nucleotide sequence
of SEQ ID NO: 1, and a DNA fragment containing the nucleotide sequence of SEQ ID NO:
1 as a template. When a DNA fragment having a length of about 300 bp is used as the
probe, the hybridization conditions for washing include, for example, 50°C, 2 x SSC
and 0.1% SDS.
[0042] The substitution, deletion, insertion, or addition of nucleotides as described above
also includes mutations which naturally occur (mutant or variant), for example, due
to variety in the species or genus of bacterium, and which contains the alcohol dehydrogenase.
[0043] A wild-type alcohol dehydrogenase may be subject to metal catalyzed oxidatation.
Although such a wild-type alcohol dehydrogenase can be used, a mutant alcohol dehydrogenase
which is resistant to aerobic inactivation is preferable in the present invention.
The phrase "mutant alcohol dehydrogenase which is resistant to aerobic inactivation"
means that the mutant alcohol dehydrogenase maintains its activity under aerobic conditions,
or the activity is reduced by a negligible amount compared to the wild-type alcohol
dehydrogenase.
[0044] In case of the
adhE gene of
E.
coli, the wild-type alcohol dehydrogenase comprises the amino acid sequence set forth in
SEQ ID NO: 2. An example of a mutation in alcohol dehydrogenase of SEQ ID NO: 2 which
results in the protein being resistant to aerobic inactivation is replacement of the
glutamic acid residue at position 568 with a lysine residue. However, introduction
of a mutation into the
adhE gene, for example at position 568 in SEQ ID NO: 2, may lead to delay of growth in
a liquid medium containing ethanol as a carbon source, and in such a case, it is preferable
that the mutant alcohol dehydrogenase have at least one additional mutation which
is able to improve the growth of the bacterium in a liquid medium which contains ethanol
as the sole carbon source. For example, the growth of
E.
coli is improved when the glutamic acid residue at position 568 in the alcohol dehydrogenase
of SEQ ID NO: 2 is replaced by another amino acid residue by introducing an additional
mutation selected from the group consisting of:
- A) replacement of the glutamic acid residue at position 560 in SEQ ID NO: 2 with another
amino acid residue, e.g., a lysine residue;
- B) replacement of the phenylalanine residue at position 566 in SEQ ID NO: 2 with another
amino acid residue, e.g., a valine residue;
- C) replacement of the glutamic acid residue, the methionine residue, the tyrosine
residue, the isoleucine residue, and the alanine residue at positions 22, 236, 461,
554, and 786, respectively, in SEQ ID NO: 2 with other amino acid residues, e.g.,
a glycine residue, a valine residue, a cysteine residue, a serine residue, and a valine
residue, respectively; and
- D) combinations thereof.
[0045] The reference to position numbers in a sequence, for example, the phrase "amino acid
residues at positions 22, 236, 554, 560, 566, 568 and 786" refers to positions of
these residues in the amino acid sequence of the wild-type AdhE from
E. coli. However, the position of an amino acid residue may change. For example, if an amino
acid residue is inserted at the N-terminus portion, the amino acid residue inherently
located at position 22 becomes position 23. In such a case, the amino acid residue
at original position 22 is the amino acid residue at position 22 in the present invention.
[0046] The mutant AdhE may include deletion, substitution, insertion, or addition of one
or several amino acids at one or a plurality of positions other than positions identified
in A) to C) above, provided that the AdhE activity is not lost or reduced.
[0047] The mutant AdhE and mutant
adhE gene according to the present invention can be obtained from the wild-type
adhE gene, for example, by site-specific mutagenesis using ordinary methods, such as PCR
(polymerase chain reaction; refer to
White, T.J. et al., Trends Genet., 5, 185 (1989)) utilizing primers prepared based on the nucleotide sequence of the gene.
[0049] In the present invention, a bacterial strain used for producing an L-amino acid is
modified so that expression of the
adhE gene is controlled by a non-native promoter, i.e., a promoter that does not control
the expression of the
adhE gene in a wild-type strain. Such modification can be achieved by replacing the native
promoter of the
adhE gene on the chromosome with a non-native promoter which functions under an aerobic
cultivation condition so that the
adhE gene is operably linked with the non-native promoter. As a non-native promoter which
functions under aerobic cultivation conditions, any promoter which can express the
adhE gene above a certain level under aerobic cultivation conditions may be used. With
reference to the level of the AdhE protein in the present invention, the activity
of alcohol dehydrogenase in the cell free extract measured according to the method
by
Clark and Cronan (J. Bacteriol. 141 177-183 (1980)) should be 1.5 units or more, preferably 5 units or more, and more preferably 10
units or more, per mg of protein. Aerobic cultivation conditions can be those usually
used for cultivation of bacteria in which oxygen is supplied by methods such as shaking,
aeration and agitation. Specifically, any promoter which is known to express a gene
under aerobic cultivation conditions can be used. For example, promoters of the genes
involved in glycosis, the pentose phosphate pathway, TCA cycle, amino acid biosynthetic
pathways, etc. can be used. In addition, the P
tac promoter, the
lac promoter, the
trp promoter, the
trc promoter, the P
R, or the P
L promoters of lambda phage are all known to be strong promoters which function under
aerobic cultivation conditions, and are preferably used.
[0050] The use of a non-native promoter can be combined with the multiplication of gene
copies. For example, inserting the
adhE gene operably linked with a non-native promoter into a vector that is able to function
in a bacterium of the
Enterobacteriaceae family and introducing the vector into the bacterium increases the copy number of
the gene in a cell. Preferably, low-copy vectors are used. Examples of low-copy vectors
include, but are not limited to, pSC101, pMW 118, pMW119, and the like. The term "low
copy vector" is used for vectors, the copy number of which is up to 5 copies per cell.
Increasing the copy number of the
adhE gene can also be achieved by introducing multiple copies of the gene into the chromosomal
DNA of the bacterium by, for example, homologous recombination, Mu integration, and
the like. Homologous recombination is carried out using a sequence which is present
in multiple copies as targets on the chromosomal DNA. Sequences having multiple copies
on the chromosomal DNA include, but are not limited to, repetitive DNA, or inverted
repeats existing at the end of a transposable element. Also, as disclosed in
U.S. Patent No. 5,595,889, it is possible to incorporate the
adhE gene into a transposon, and allow it to be transferred to introduce multiple copies
of the gene into the chromosomal DNA. In these instances, the
adhE gene can be placed under the control of a promoter which functions under aerobic
cultivation conditions. Alternatively, the effect of a promoter can be enhanced by,
for example, introducing a mutation into the promoter to increase the transcription
level of a gene located downstream of the promoter. Furthermore, it is known that
the substitution of several nucleotides in the spacer between the ribosome binding
site (RBS) and the start codon, especially the sequences immediately upstream of the
start codon, profoundly affect the mRNA translatability. For example, a 20-fold range
in the expression levels was found, depending on the nature of the three nucleotides
preceding the start codon (
Gold et al., Annu. Rev. Microbiol., 35, 365-403, 1981;
Hui et al., EMBO J., 3, 623-629, 1984). Previously, it was shown that the
rhtA23 mutation is an A-for-G substitution at the -1 position relative to the ATG start
codon (
ABSTRACTS of 17th International Congress of Biochemistry and Molecular Biology in
conjugation with 1997 Annual Meeting of the American Society for Biochemistry and
Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457). Therefore, it may be suggested that the
rhtA23 mutation enhances
rhtA gene expression and, as a consequence, increases resistance to threonine, homoserine,
and some other substances transported out of cells.
[0051] Moreover, it is also possible to introduce a nucleotide substitution into a promoter
region of the
adhE gene on the bacterial chromosome, which results in stronger promoter function. The
alteration of the expression control sequence can be performed, for example, in the
same manner as the gene substitution using a temperature-sensitive plasmid, as disclosed
in International Patent Publication
WO 00/18935 and Japanese Patent Application Laid-Open No.
1-215280.
[0052] In the present invention, "L-amino acid-producing bacterium" means a bacterium which
has an ability to produce and secrete an L-amino acid into a medium, when the bacterium
is cultured in the medium. The L-amino acid-producing ability may be imparted or enhanced
by breeding. The term "L-amino acid-producing bacterium" as used herein also means
a bacterium which is able to produce and cause accumulation of an L-amino acid in
a culture medium in an amount larger than a wild-type or parental strain of the bacterium,
for example,
E. coli, such as
E. coli K-12, and preferably means that the bacterium is able to cause accumulation in a
medium of an amount not less than 0.5 g/L, more preferably not less than 1.0 g/L of
the target L-amino acid. The term "L-amino acid" includes L-alanine, L-arginine, L-asparagine,
L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine,
L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,
L-tryptophan, L-tyrosine, and L-valine. L-threonine, L-lysine, L-histidine, L-phenylalanine,
L-arginine, L-tryptophan, L-glutamic acid, and L-leucine are particularly preferred.
[0053] The
Enterobacteriaceae family includes bacteria belonging to the genera
Escherichia, Enterobacter, Erwinia, Klebsiella, Pantoea, Photorhabdus, Providencia,
Salmonella, Serratia, Shigella, Morganella, Yersinia, etc.. Specifically, those classified into the
Enterobacteriaceae family according to the taxonomy used by the NCBI (National Center for Biotechnology
Information) database (http://www.ncbi.nlm.nih.gov/TaxonomyBrowser/wwwtax.cgi?id=91347)
can be used. A bacterium belonging to the genus
Escherichia or
Pantoea is preferred. The phrase "a bacterium belonging to the genus
Escherichia" means that the bacterium is classified into the genus
Escherichia according to the classification known to a person skilled in the art of microbiology.
Examples of a bacterium belonging to the genus
Escherichia as used in the present invention include, but are not limited to,
Escherichia coli (
E. coli).
[0055] The bacterium belonging to the genus
Pantoea means that the bacterium is classified into the genus
Pantoea according to the classification known to a person skilled in the art of microbiology.
Some species of
Enterobacter agglomerans have been recently re-classified into
Pantoea agglomerans, Pantoea ananatis, Pantoea stewartii, or the like, based on the nucleotide sequence analysis of 16S rRNA etc. (
Int. J. Syst. Bacteriol., 43, 162-173 (1993)).
[0056] The bacterium of the present invention encompasses a strain of the
Enterobacteriaceae family which has an ability to produce an L-amino acid and has been modified so that
the gene encoding an alcohol dehydrogenase is expressed under the control of a promoter
which functions under aerobic cultivation conditions. In addition, the bacterium of
the present invention encompasses a strain of the
Enterobacteriaceae family which has an ability to produce an L-amino acid and does not have a native
activity of alcohol dehydrogenase, but has been transformed with a DNA fragment encoding
alcohol dehydrogenase.
[0057] In the present invention, the amount of accumulated L-amino acid, for example, L-threonine,
L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan, L-glutamic acid,
or L-leucine, can be significantly increased in a culture medium containing ethanol
as a carbon source as a result of expressing the gene encoding an alcohol dehydrogenase
under the control of a promoter which functions under aerobic cultivation conditions.
L-amino acid-producing bacteria
[0058] As a bacterium of the present invention which is modified to have mutant alcohol
dehydrogenase of the present invention, bacteria which are able to produce either
an aromatic or a non-aromatic L-amino acids may be used.
[0059] The bacterium of the present invention can be obtained by introducing the gene encoding
the mutant alcoholdehydrogenase of the present invention in a bacterium which inherently
has the ability to produce L-amino acids. Alternatively, the bacterium of present
invention can be obtained by imparting the ability to produce L-amino acids to a bacterium
already having the mutant alcohol dehydrogenase.
L-threonine-producing bacteria
[0060] Examples of parent strains which can be used to derive the L-threonine-producing
bacteria of the present invention include, but are not limited to, strains belonging
to the genus
Escherichia, such as
E. coli TDH-6/pVIC40 (VKPM B-3996) (
U.S. Patent No. 5, 175, 107,
U.S. Patent No. 5,705,371),
E. coli 472T23/pYN7 (ATCC 98081) (
U.S. Patent No.5,631,157),
E. coli NRRL-21593 (
U.S. Patent No. 5,939,307),
E. coli FERM BP-3756 (
U.S. Patent No. 5,474,918),
E. coli FERM BP-3519 and FERM BP-3520 (
U.S. Patent No. 5,376,538),
E. coli MG442 (
Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)),
E. coli VL643 and VL2055 (
EP 1149911 A), and the like.
[0061] The strain TDH-6 is deficient in the
thrC gene, as well as being sucrose-assimilative, and the
ilvA gene in this strain has a leaky mutation. This strain also has a mutation in the
rhtA gene, which imparts resistance to high concentrations of threonine or homoserine.
The strain B-3996 contains the plasmid pVIC40 which was obtained by inserting a
thrA*BC operon which includes a mutant
thrA gene into a RSF1010-derived vector. This mutant
thrA gene encodes aspartokinase homoserine dehydrogenase I which has substantially desensitized
feedback inhibition by threonine. The strain B-3996 was deposited on November 19,
1987 in the All-Union Scientific Center of Antibiotics (Russia, 117105 Moscow, Nagatinskaya
Street, 3-A) under the accession number RIA 1867. The strain was also deposited in
the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545
Moscow, 1 Dorozhny proezd, 1) on April 7, 1987 under the accession number VKPM B-3996.
[0062] E. coli VKPM B-5318 (
EP 0593792B) also may be used as a parent strain to derive L-threonine-producing bacteria of
the present invention. The strain B-5318 is prototrophic with regard to isoleucine,
and a temperature-sensitive lambda-phage C 1 repressor and P
R promoter replaces the regulatory region of the threonine operon in the plasmid pVIC40
harbored by the strain. The strain VKPM B-5318 was deposited in the Russian National
Collection of Industrial Microorganisms (VKPM) on May 3, 1990 under accession number
of VKPM B-5318.
[0063] Preferably, the bacterium of the present invention is additionally modified to enhance
expression of one or more of the following genes:
- the mutant thrA gene which codes for aspartokinase-homoserine dehydrogenase I resistant
to feed back inhibition by threonine;
- the thrB gene which codes for homoserine kinase;
- the thrC gene which codes for threonine synthase;
- the rhtA gene which codes for a putative transmembrane protein;
- the asd gene which codes for aspartate-β-semialdehyde dehydrogenase; and
- the aspC gene which codes for aspartate aminotransferase (aspartate transaminase);
[0064] The
thrA gene which encodes aspartokinase-homoserine dehydrogenase I of
Escherichia coli has been elucidated (nucleotide positions 337 to 2799, GenBank accession no.NC_000913.2,
gi: 49175990). The
thrA gene is located between the
thrL and
thrB genes on the chromosome of
E.
coli K-12. The
thrB gene which encodes homoserine kinase of
Escherichia coli has been elucidated (nucleotide positions 2801 to 3733, GenBank accession NC_000913.2,
gi: 49175990). The
thrB gene is located between the
thrA and
thrC genes on the chromosome
of E. coli K-12. The
thrC gene which encodes threonine synthase of
Escherichia coli has been elucidated (nucleotide positions 3734 to 5020, GenBank accession NC_000913.2,
gi: 49175990). The
thrC gene is located between the
thrB gene and the
yaaX open reading frame on the chromosome of
E.
coli K-12. All three genes function as a single threonine operon. To enhance expression
of the threonine operon, the attenuator region which affects the transcription is
desirably removed from the operon (
WO2005/049808,
WO2003/097839).
[0065] A mutant
thrA gene which codes for aspartokinase homoserine dehydrogenase I resistant to feedback
inhibition by threonine, as well as the
thrB and
thrC genes can be obtained as one operon from the well-known plasmid pVIC40, which is
present in the threonine producing
E. coli strain VKPM B-3996. Plasmid pVIC40 is described in detail in
U.S. Patent No. 5,705,371.
[0066] The
rhtA gene is located at 18 min on the
E. coli chromosome close to the
glnHPQ operon, which encodes components of the glutamine transport system. The
rhtA gene is identical to ORF1 (
ybiF gene, nucleotide positions 764 to 1651, GenBank accession number AAA218541, gi:440181),
and is located between the
pexB and
ompX genes. The DNA sequence expressing a protein encoded by the ORF1 has been designated
the
rhtA gene (rht: resistance to homoserine and threonine). Also, it is known that the
rhtA23 mutation is an A-for-G substitution at position -1 with respect to the ATG start
codon (
ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology
in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular
Biology, San Francisco, California August 24-29, 1997, abstract No. 457,
EP 1013765 A). Hereinafter, the
rhtA23 mutation is marked as
rhtA*.
[0067] The
asd gene of
E.
coli has already been elucidated (nucleotide positions 3572511 to 3571408, GenBank accession
NC_000913.1, gi:16131307), and can be obtained by PCR (polymerase chain reaction;
refer to
White, T.J. et al., Trends Genet., 5, 185 (1989)) utilising primers prepared based on the nucleotide sequence of the gene. The
asd genes of other microorganisms can be obtained in a similar manner.
[0068] Also, the
aspC gene of
E. coli has already been elucidated (nucleotide positions 983742 to 984932, GenBank accession
NC_000913.1, gi:16128895), and can be obtained by PCR. The
aspC genes of other microorganisms can be obtained in a similar manner.
L-lysine-producing bacteria
[0069] Examples of L-lysine-producing bacteria belonging to the genus
Escherichia include mutants having resistance to an L-lysine analogue. The L-lysine analogue
inhibits growth of bacteria belonging to the genus
Escherichia, but this inhibition is fully or partially desensitized when L-lysine is present in
the medium. Examples of the L-lysine analogue include, but are not limited to, oxalysine,
lysine hydroxamate, S-(2-aminoethyl)-L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam,
and so forth. Mutants having resistance to these lysine analogues can be obtained
by subjecting bacteria belonging to the genus
Escherichia to a conventional artificial mutagenesis treatment. Specific examples of bacterial
strains useful for producing L-lysine include
Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see
U.S. Patent No. 4,346,170) and
Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine
is desensitized.
[0070] The strain WC 196 may be used as an L-lysine producing bacterium of
Escherichia coli. This bacterial strain was bred by conferring AEC resistance to the strain W3110,
which was derived from
Escherichia coli K-12. The resulting strain was designated
Escherichia coli AJ13069 and was deposited at the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology (currently National Institute of Advanced
Industrial Science and Technology, International Patent Organism Depositary, Tsukuba
Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan) on December
6, 1994 and received an accession number of FERM P-14690. Then, it was converted to
an international deposit under the provisions of the Budapest Treaty on September
29, 1995, and received an accession number of FERM BP-5252 (
U.S. Patent No. 5,827,698).
[0071] Examples of parent strains which can be used to derive L-lysine-producing bacteria
of the present invention also include strains in which expression of one or more genes
encoding an L-lysine biosynthetic enzyme are enhanced. Examples of such genes include,
but are not limited to, genes encoding dihydrodipicolinate synthase (
dapA), aspartokinase (
lysC), dihydrodipicolinate reductase (
dapB), diaminopimelate decarboxylase (
lysA), diaminopimelate dehydrogenase (
ddh) (
U.S. Patent No. 6,040,160), phosphoenolpyruvate carboxylase (
ppc), aspartate semialdehyde dehydrogenase (
asd), and aspartase (
aspA) (
EP 1253195 A). In addition, the parent strains may have an increased level of expression of the
gene involved in energy efficiency (
cyo) (
EP 1170376 A), the gene encoding nicotinamide nucleotide transhydrogenase (
pntAB) (
U.S. Patent No. 5,830,716), the
ybjE gene (
W02005/073390), or combinations thereof.
[0072] Examples of parent strains for deriving L-lysine-producing bacteria of the present
invention also include strains having decreased or eliminated activity of an enzyme
that catalyzes a reaction for generating a compound other than L-lysine by branching
off from the biosynthetic pathway of L-lysine. Examples of the enzymes that catalyze
a reaction for generating a compound other than L-lysine by branching off from the
biosynthetic pathway of L-lysine include homoserine dehydrogenase, lysine decarboxylase
(
U.S. Patent No. 5,827,698), and the malic enzyme (
W02005/010175).
[0073] Examples of L-lysine producing strains include
E. coli WC196ΔcadAΔldc/pCABD2 (
WO2006/078039). This strain was obtained by introducing the plasmid pCABD2, which is disclosed
in
U.S. Patent No. 6,040,160, into the strain WC196 with the disrupted
cadA and
ldcC genes, which encode lysine decarboxylase. The plasmid pCABD2 contains the
dapA gene of
E. coli coding for a dihydrodipicolinate synthase having a mutation which desensitizes feedback
inhibition by L-lysine, the
lysC gene of
E. coli coding for aspartokinase III having a mutation which desensitizes feedback inhibition
by L-lysine, the
dapB gene
E. coli coding for a dihydrodipicolinate reductase, and the
ddh gene of Corynebacterium glutamicum coding for diaminopimelate dehydrogenase..
L-cysteine-producing bacteria
[0074] Examples of parent strains which can be used to derive L-cysteine-producing bacteria
of the present invention include, but are not limited to, strains belonging to the
genus
Escherichia, such as
E. coli JM15 which is transformed with different
cysE alleles coding for feedback-resistant serine acetyltransferases (
U.S. Patent No. 6,218,168, Russian patent application
2003121601);
E. coli W3110 which over-expresses genes which encode proteins suitable for secreting substances
toxic for cells (
U.S. Patent No. 5,972,663);
E. coli strains having lowered cysteine desulfohydrase activity (
JP11155571A2);
E. coli W3110 with increased activity of a positive transcriptional regulator for cysteine
regulon encoded by the
cysB gene (
WO0127307A1), and the like.
L-leucine-producing bacteria
[0075] Examples of parent strains which can be used to derive L-leucine-producing bacteria
of the present invention include, but are not limited to, strains belonging to the
genus
Escherichia, such as
E. coli strains resistant to leucine (for example, the strain 57 (VKPM B-7386,
U.S. Patent No. 6,124,121)) or leucine analogs including β-2-thienylalanine, 3-hydroxyleucine, 4-azaleucine,
5,5,5-trifluoroleucine (
JP 62-34397 B and
JP 8-70879 A);
E. coli strains obtained by the genetic engineering methods such as those described in
WO96/06926;
E. coli H-9068 (
JP 8-70879 A), and the like.
[0076] The bacterium of the present invention may be improved by enhancing the expression
of one or more genes involved in L-leucine biosynthesis. Examples include genes of
the
leuABCD operon, which are preferably represented by a mutant
leuA gene coding for isopropylmalate synthase which is not subject to feedback inhibition
by L-leucine (
US Patent 6,403,342). In addition, the bacterium of the present invention may be improved by enhancing
the expression of one or more genes coding for proteins which excrete L-amino acids
from the bacterial cell. Examples of such genes include the b2682 and b2683 genes
(
ygaZH genes) (
EP 1239041 A2).
L-histidine-producing bacteria
[0077] Examples of parent strains which can be used to derive L-histidine-producing bacteria
of the present invention include, but are not limited to, strains belonging to the
genus
Escherichia, such as
E. coli strain 24 (VKPM B-5945, RU2003677),
E. coli strain 80 (VKPM B-7270, RU2119536),
E. coli NRRL B-12116 - B12121 (
U.S. Patent No. 4,388,405),
E. coli H-9342 (FERM BP-6675) and H-9343 (FERM BP-6676) (
U.S. Patent No. 6,344,347),
E. coli H-9341 (FERM BP-6674) (
EP1085087),
E. coli AI80/pFM201 (
U,S. Patent No. 6,258,554), and the like.
[0078] Examples of parent strains which can be used to derive L-histidine-producing bacteria
of the present invention also include strains in which expression of one or more genes
encoding an L-histidine biosynthetic enzyme are enhanced. Examples of such genes include
genes encoding ATP phosphoribosyltransferase (
hisG), phosphoribosyl AMP cyclohydrolase (
hisI), phosphoribosyl-ATP pyrophosphohydrolase (
hisIE), phosphoribosylformimino-5-aminoimidazole carboxamide ribotide isomerase (
hisA), amidotransferase (
hisH), histidinol phosphate aminotransferase (
hisC), histidinol phosphatase (
hisB), histidinol dehydrogenase (
hisD), and so forth.
[0079] It is known that the L-histidine biosynthetic enzymes encoded by
hisG and
hisBHAFI are inhibited by L-histidine, and therefore an L-histidine-producing ability can
also be efficiently enhanced by introducing a mutation into any of these genes which
confer resistance to the feedback inhibition into enzymes encoded by the genes (Russian
Patent Nos.
2003677 and
2119536).
[0080] Specific examples of strains having an L-histidine-producing ability include
E. coli FERM P-5038 and 5048 which have been transformed with a vector carrying a DNA encoding
an L-histidine-biosynthetic enzyme (
JP 56-005099 A),
E. coli strains transformed with
rht, a gene for an amino acid-exporter (
EP1016710A),
E. coli 80 strain imparted with sulfaguanidine, DL-1,2,4-triazole-3-alanine, and streptomycin-resistance
(VKPM B-7270, Russian Patent No.
2119536), and so forth.
L-glutamic acid-producing bacteria
[0081] Examples of parent strains which can be used to derive L-glutamic acid-producing
bacteria of the present invention include, but are not limited to, strains belonging
to the genus
Escherichia, such as
E. coli VL334thrC
+(
EP 1172433).
E. coli VL334 (VKPM B-1641) is an L-isoleucine and L-threonine auxotrophic strain having
mutations in the
thrC and
ilvA genes (
U.S. Patent No. 4,278,765). A wild-type allele of the
thrC gene was transferred using general transduction with a bacteriophage P1 grown on
the wild-type
E. coli strain K12 (VKPM B-7) cells. As a result, an L-isoleucine auxotrophic strain VL334thrC
+ (VKPM B-8961), which is able to produce L-glutamic acid, was obtained.
[0082] Examples of parent strains which can be used to derive the L-glutamic acid-producing
bacteria of the present invention include, but are not limited to, strains which are
deficient in α-ketoglutarate dehydrogenase activity, or strains in which expression
of one or more genes encoding an L-glutamic acid biosynthetic enzyme are enhanced.
Examples of such genes include genes encoding glutamate dehydrogenase (
gdh), glutamine synthetase (
glnA), glutamate synthetase (
gltAB), isocitrate dehydrogenase (
icdA), aconitate hydratase (
acnA, acnB), citrate synthase (
gltA), phosphoenolpyruvate carboxylase (
ppc), pyruvate dehydrogenase (
aceEF, lpdA), pyruvate kinase (
pykA, pykF), phosphoenolpyruvate synthase (
ppsA), enolase (
eno), phosphoglyceromutase (
pgmA, pgmI), phosphoglycerate kinase (
pgk), glyceraldehyde-3-phophate dehydrogenase (
gapA), triose phosphate isomerase (
tpiA), fructose bisphosphate aldolase (
fbp), phosphofructokinase (
pfkA, pfkB), glucose phosphate isomerase (
pgi), and so forth.
[0083] Examples of strains which have been modified so that expression of the citrate synthetase
gene and/or the phosphoenolpyruvate carboxylase gene are reduced, and/or are deficient
in α-ketoglutarate dehydrogenase activity include those disclosed in
EP1078989A,
EP955368A, and
EP952221A.
[0084] Examples of parent strains which can be used to derive the L-glutamic acid-producing
bacteria of the present invention also include strains having decreased or eliminated
activity of an enzyme that catalyzes synthesis of a compound other than L-glutamic
acid by branching off from an L-glutamic acid biosynthesis pathway. Examples of such
enzymes include isocitrate lyase (
aceA), α-ketoglutarate dehydrogenase (
sucA), phosphotransacetylase (
pta), acetate kinase (
ack), acetohydroxy acid synthase (
ilvG), acetolactate synthase (
ilvI), formate acetyltransferase (
pfl), lactate dehydrogenase (
ldh), and glutamate decarboxylase (
gadAB). Bacteria belonging to the genus
Escherichia deficient in α-ketoglutarate dehydrogenase activity or having a reduced α-ketoglutarate
dehydrogenase activity and methods for obtaining them are described in
U.S. Patent Nos. 5,378,616 and
5,573,945. Specifically, these strains include the following:
E. coli W3110sucA::KmR
E. coli AJ12624 (FERM BP-3853)
E. coli AJ12628 (FERM BP-3854)
E. coli AJ12949 (FERM BP-4881)
E. coli W3110sucA::KmR is obtained by disrupting the α-ketoglutarate dehydrogenase gene (hereinafter referred
to as "sucA gene") of E. coli W3110. This strain is completely deficient in the α-ketoglutarate dehydrogenase activity.
[0085] Other examples of L-glutamic acid-producing bacteria include those which belong to
the genus
Escherichia and have resistance to an aspartic acid antimetabolite. These strains can also be
deficient in the α-ketoglutarate dehydrogenase activity and include, for example,
E. coli AJ13199 (FERM BP-5807) (
U.S. Patent No. 5.908,768), FFRM P-12379, which additionally has a low L-glutamic acid decomposing ability
(
U.S. Patent No. 5,393,671), AJ13138 (FERM BP-5565) (
U.S. Patent No. 6,110,714), and the like.
[0086] Examples of L-glutamic acid-producing bacteria, include mutant strains belonging
to the genus
Pantoea which are deficient in α-ketoglutarate dehydrogenase activity or have decreased α-ketoglutarate
dehydrogenase activity, and can be obtained as described above. Such strains include
Pantoea ananatis AJ13356. (
U.S. Patent No. 6,331,419).
Pantoea ananatis AJ13356 was deposited at the National Institute of Bioscience and Human-Technology,
Agency of Industrial Science and Technology, Ministry of International Trade and Industry
(currently, National Institute of Advanced Industrial Science and Technology, International
Patent Organism Depositary, Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken,
305-8566, Japan) on February 19, 1998 under an accession number of FERM P-16645. It
was then converted to an international deposit under the provisions of Budapest Treaty
on January 11, 1999 and received an accession number of FERM BP-6615.
Pantoea ananatis AJ13356 is deficient in the α-ketoglutarate dehydrogenase activity as a result of
disruption of the αKGDH-E1 subunit gene (
sucA). The above strain was identified as
Enterobacter agglomerans when it was isolated and deposited as
Enterobacter agglomerans AJ13356. However, it was recently re-classified as
Pantoea ananatis on the basis of nucleotide sequencing of 16S rRNA and so forth. Although AJ13356
was deposited at the aforementioned depository as
Enterobacter agglomerans, for the purposes of this specification, they are described as
Pantoea ananatis.
L-phenylalanine-producing bacteria
[0087] Examples of parent strains which can be used to derive L-phenylalanine-producing
bacteria of the present invention include, but are not limited to, strains belonging
to the genus
Escherichia, such as
E. coli AJ12739 (tyrA::Tn10, tyrR) (VKPM B-8197),
E. coli HW1089 (ATCC 55371) harboring the mutant
pheA34 gene (
U.S. Patent No. 5,354,672),
E. coli MWEC101-b (
KR8903681),
E. coli NRRL B-12141, NRRL B-12145, NRRL B-12146 and NRRL B-12147 (
U.S. Patent No. 4,407,952). Also, as a parent strain,
E. coli K-12 [W3110 (tyrA)/pPHAB (FERM BP-3566),
E. coli K-12 [W3110 (tyrA)/pPHAD] (FERM BP-12659),
E. coli K-12 [W3110 (tyrA)/pPHATerm] (FERM BP-12662) and
E. coli K-12 [W3110 (tyrA)/pBR-aroG4, pACMAB] named as AJ 12604 (FERM BP-3579) may be used
(
EP 488424 B1). Furthermore, L-phenylalanine producing bacteria belonging to the genus
Escherichia with an enhanced activity of the protein encoded by the
yedA gene or the
yddG gene may also be used (
U.S. patent applications 2003/0148473 A1 and
2003/0157667 A1).
L-tryptophan-producing bacteria
[0088] Examples of parent strains which can be used to derive the L-tryptophan-producing
bacteria of the present invention include, but are not limited to, strains belonging
to the genus
Escherichia, such as
E. coli JP4735/pMU3028 (DSM10122) and JP6015/pMU91 (DSM10123) which is deficient in tryptophanyl-tRNA
synthetase encoded by the mutant
trpS gene (
U.S. Patent No. 5,756,345),
E. coli SV164 (pGH5) having a
serA allele encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition
by serine and a
trpE allele encoding anthranilate synthase which is not subject to feedback inhibition
by tryptophan (
U.S. Patent No. 6,180,373),
E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6(pGX50)aroP (NRRL B-12264) which is deficient
in the enzyme tryptophanase (
U.S. Patent No. 4,371,614),
E.
coli AGX17/pGX50,pACKG4-pps in which a phosphoenolpyruvate-producing ability is enhanced
(
WO9708333,
U.S. Patent No. 6,319,696), and the like. L-tryptophan-producing bacteria belonging to the genus
Escherichia which have enhanced activity of the protein encoded by the
yedA or
yddG genes may also be used (
U.S. patent applications 2003/0148473 A1 and
2003/0157667 A1).
[0089] Examples of parent strains which can be used to derive the L-tryptophan-producing
bacteria of the present invention also include strains in which one or more activities
are enhanced of the following enzymes: anthranilate synthase (
trpE), phosphoglycerate dehydrogenase (
serA), and tryptophan synthase (
trpAB). The anthranilate synthase and phosphoglycerate dehydrogenase are both subject to
feedback inhibition by L-tryptophan and L-serine, therefore a mutation desensitizing
the feedback inhibition may be introduced into these enzymes. Specific examples of
strains having such a mutation include
E. coli SV164 which harbors desensitized anthranilate synthase and a transformant strain
obtained by introducing into
E. coli SV164 the plasmid pGH5 (
WO 94/08031), which contains a mutant
serA gene encoding feedback-desensitized phosphoglycerate dehydrogenase.
[0090] Examples of parent strains which can be used to derive the L-tryptophan-producing
bacteria of the present invention also include strains which have been transformed
with the tryptophan operon containing a gene encoding desensitized anthranilate synthase
(
JP 57-71397 A,
JP 62-244382 A,
U.S. Patent No. 4,371,614). Moreover, L-tryptophan-producing ability may be imparted by enhancing expression
of a gene which encodes tryptophan synthase, among tryptophan operons (
trpBA). Tryptophan synthase consists of α and β subunits which are encoded by the
trpA and
trpB genes, respectively. In addition, L-tryptophan-producing ability may be improved
by enhancing expression of the isocitrate lyase-malate synthase operon (
WO2005/103275).
L-proline-producing bacteria
[0091] Examples of parent strains which can be used to derive L-proline-producing bacteria
of the present invention include, but are not limited to, strains belonging to the
genus
Escherichia, such as
E. coli 702ilvA (VKPM B-8012) which is deficient in the
ilvA gene and is able to produce L-proline (
EP 1172433). The bacterium of the present invention may be improved by enhancing the expression
of one or more genes involved in L-proline biosynthesis. Examples of such genes include
the
proB gene coding for glutamate kinase which is desensitized to feedback inhibition by
L-proline (
DE Patent 3127361). In addition, the bacterium of the present invention may be improved by enhancing
the expression of one or more genes coding for proteins responsible for secreting
L-amino acids from the bacterial cell. Such genes are exemplified by the b2682 and
b2683 genes (
ygaZH genes) (
EP 1239041 A2).
L-arginine-producing bacteria
[0093] Examples of parent strains which can be used to derive L-arginine-producing bacteria
of the present invention include, but are not limited to, strains belonging to the
genus
Escherichia, such as
E. coli strain 237 (VKPM B-7925) (
U.S. Patent Application 2002/058315 A1) and derivatives thereof harboring mutant N-acetylglutamate synthase (Russian Patent
Application No.
2001112869),
E. coli strain 382 (VKPM B-7926) (
EP1170358A1), an arginine-producing strain transformed with the
argA gene encoding N-acetylglutamate synthetase (
EP1170361A1), and the like.
[0094] Examples of parent strains which can be used to derive L-arginine producing bacteria
of the present invention also include strains in which expression of one or more genes
encoding an L-arginine biosynthetic enzyme are enhanced. Examples of such genes include
genes encoding N-acetylglutamyl phosphate reductase (
argC), ornithine acetyl transferase (
argJ), N-acetylglutamate kinase (
argB), acetylornithine transaminase (
argD), ornithine carbamoyl transferase (
argF), argininosuccinic acid synthetase (
argG), argininosuccinic acid lyase (
argH), carbamoyl phosphate synthetase (
carAB), and so forth.
L-valine-producing bacteria
[0095] Example of parent strains which can be used to derive L-valine-producing bacteria
of the present invention include, but are not limited to, strains which have been
modified to overexpress the
ilvGMEDA operon (
U.S. Patent No. 5,998,178). It is desirable to remove the region of the
ilvGMEDA operon responsible for attenuation so that the produced L-valine cannot attenuate
expression of the operon. Furthermore, the
ilvA gene in the operon is desirably disrupted so that threonine deaminase activity is
decreased.
[0096] Examples of parent strains which can be used to derive L-valine-producing bacteria
of the present invention also include mutants of amino-acyl t-RNA synthetase (
U.S. Patent No. 5,658,766). For example,
E. coli VL1970, which has a mutation in the
ileS gene encoding isoleucine tRNA synthetase, can be used.
E. coli VL1970 has been deposited in the Russian National Collection of Industrial Microorganisms
(VKPM) (Russia, 117545 Moscow, 1 Dorozhny Proezd, 1) on June 24, 1988 under accession
number VKPM B-4411.
[0097] Furthermore, mutants requiring lipoic acid for growth and/or lacking H
+-ATPase can also be used as parent strains (
WO96/06926).
L-isoleucine-producing bacteria
[0098] Examples of parent strains which can be used to derive L-isoleucine producing bacteria
of the present invention include, but are not limited to, mutants having resistance
to 6-dimethylaminopurine (
JP 5-304969 A), mutants having resistance to an isoleucine analogue such as thiaisoleucine and
isoleucine hydroxamate, and mutants additionally having resistance to DL-ethionine
and/or arginine hydroxamate (
JP 5-130882 A). In addition, recombinant strains transformed with genes encoding proteins involved
in L-isoleucine biosynthesis, such as threonine deaminase and acetohydroxate synthase,
can also be used as parent strains (
JP 2-458 A,
FR 0356739, and
U.S. Patent No. 5,998,178).
[0099] The method for producing an L-amino acid of the present invention includes the steps
of cultivating the bacterium of the present invention in a culture medium, allowing
L-amino acid to accumulate in the culture medium, and collecting L-amino acid from
the culture medium. Furthermore, the method of present invention includes a method
for producing L-threonine, L-lysine, L-histidine, L-phenylalanine, L-arginine, L-tryptophan,
L-glutamic acid, or L-leucine, including the steps of cultivating the bacterium of
the present invention in a culture medium, allowing L-threonine, L-lysine, L-histidine,
L-phenylalanine, L-arginine, L-tryptophan, L-glutamic acid, or L-leucine to accumulate
in the culture medium, and collecting L-threonine, L-lysine, L-histidine, L-phenylalanine,
L-arginine, L-tryptophan, L-glutamic acid, or L-leucine from the culture medium.
[0100] In the present invention, the cultivation, collection, and purification of L-amino
acids from the medium and the like may be performed by conventional fermentation methods
wherein an L-amino acid is produced using a bacterium.
[0101] The culture medium may be either synthetic or natural, so long as the medium includes
a carbon source, a nitrogen source, minerals, and if necessary, appropriate amounts
of nutrients which the bacterium requires for growth. The carbon source may include
various carbohydrates such as glucose and sucrose, various organic acids and alcohols,
such as ethanol. According to the present invention ethanol can be used as the sole
carbon source or mixed with carbohydrates, such as glucose and sucrose. As the nitrogen
source, various ammonium salts such as ammonia and ammonium sulfate, other nitrogen
compounds such as amines, a natural nitrogen source such as peptone, soybean-hydrolysate,
and digested fermentative microorganisms can be used. As minerals, potassium monophosphate,
magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium chloride,
and the like, can be used. As vitamins, thiamine, yeast extract, and the like may
be used. Additional nutrients may be added to the medium, if necessary. For example,
if the bacterium requires an L-amino acid for growth (L-amino acid auxotrophy), a
sufficient amount of the L-amino acid may be added to the cultivation medium.
[0102] The cultivation is preferably performed under aerobic conditions such as a shaking
culture, and stirring culture with aeration, at a temperature of 20 to 40°C, preferably
30 to 38°C. The pH of the culture is usually between 5 and 9, preferably between 6.5
and 7.2. The pH of the culture can be adjusted with ammonia, calcium carbonate, various
acids, various bases, and buffers. Usually, a 1 to 5-day cultivation leads to accumulation
of the target L-amino acid in the liquid medium.
[0103] After cultivation, solids such as cells can be removed from the liquid medium by
centrifugation or membrane filtration, and then the target L-amino acid can be collected
and purified by ion-exchange, concentration, and/or crystallization methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0104]
Figure 1 shows the structure of the upstream region of the adhE gene in the chromosome of E. coli and the structure of an integrated DNA fragment containing the cat gene and a PL-tac promoter.
Figure 2 shows the alignment of the primary sequences of alcohol dehydrogenase from
Escherichia coli (ADHE_ECOLI, SEQ ID NO: 2), Shigella flexneri (Q83RN2_SHIFL, SEQ ID NO: 53), Pantoea ananatis (ADHE PANAN, SEQ ID NO: 30), Yersinia pestis (Q66AM7_YERPS, SEQ ID NO: 54), Erwinia carotovora (Q6D4R4_ERWCT, SEQ ID NO: 55), Salmonella typhimurium (P74880_SALTY, SEQ ID NO: 56), Lactobacillus plantarum (Q88RY9_LACPL, SEQ ID NO: 57) and Lactococcus lactis (086282_9LACT, SEQ ID NO: 58). The alignment was done by using the PIR Multiple Alignment
program (http://pir.georgetown.edu). The identical amino acids are marked by asterisk
(*), similar amino acids are marked by colon (:).
Figure 3 shows growth curves of modified strains grown on the minimal M9 medium containing
ethanol (2% or 3%) as a sole carbon source.
Figure 4 shows growth curves of modified strains grown on the minimal M9 medium containing
a mixture of glucose (0.1 weight %) and ethanol (0.1 volume %).
Figure 5 shows comparison of growth curves of strains having mutant adhE* gene under
control of the native promoter, or PL-tac promoter grown on the minimal M9 medium containing ethanol (2% or 3%) as a sole carbon
source.
Examples
[0105] The present invention will be more concretely explained below with reference to the
following non-limiting examples.
Example 1. Preparation of E. coli MG1655 Δtdh, rhtA*
[0106] The L-threonine producing
E. coli strain MG1655 Δtdh, rhtA* (pVIC40) was constructed by inactivation of the native
tdh gene encoding threonine dehydrogenase in
E. coli MG1655 (ATCC 700926) using the
cat gene followed by introduction of an rhtA23 mutation (
rhtA*) which confers resistance to high concentrations of threonine (>40 mg/ml) and homoserine
(>5 mg/ml). Then, the resulting strain was transformed with plasmid pVIC40 from
E. coli VKPM B-3996. The plasmid pVIC40 is described in detail in
U.S. Patent No. 5,705,371.
[0107] To replace the native
tdh gene, a DNA fragment carrying the chloramphenicol resistance marker (Cm
R) encoded by the
cat gene was integrated into the chromosome of
E.
coli MG1655 in place of the native gene by the method described by
Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration".
The recombinant plasmid pKD46 (
Datsenko, K.A., Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) with the thermosensitive replicon was used as the donor of the phage λ-derived genes
responsible for the Red-mediated recombination system.
E. coli BW25113 containing the recombinant plasmid pKD46 can be obtained from the
E.
coli Genetic Stock Center, Yale University, New Haven, USA, the accession number of which
is CGSC7630.
[0108] A DNA fragment containing a Cm
R marker encoded by the
cat gene was obtained by PCR using the commercially available plasmid pACYC184 (GenBank/EMBL
accession number X06403, "Fermentas", Lithuania) as the template, and primers P1 (SEQ
ID NO: 3) and P2 (SEQ ID NO: 4). Primer P1 contains 35 nucleotides homologous to the
5'-region of the
tdh gene introduced into the primer for further integration into the bacterial chromosome.
Primer P2 contains 32 nucleotides homologous to the 3'-region of the
tdh gene introduced into the primer for further integration into the bacterial chromosome.
[0109] PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x PCR-buffer with
25 mM MgCl
2 ("Fermentas", Lithuania), 200 µM each of dNTP, 25 pmol each of the exploited primers
and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 5 ng of the plasmid
DNA was added in the reaction mixture as a template DNA for the PCR amplification.
The temperature profile was the following: initial DNA denaturation for 5 min at 95
°C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C
for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at
72 °C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis,
extracted using "GenElute Spin Columns" (Sigma, USA), and precipitated by ethanol.
[0110] The obtained DNA fragment was used for electroporation and Red-mediated integration
into the bacterial chromosome of
E.
coli MG1655/pKD46.
[0111] MG1655/pKD46 cells were grown overnight at 30 °C in liquid LB-medium containing ampicillin
(100 µg/ml), then diluted 1:100 by SOB-medium (Yeast extract, 5 g/l; NaCl, 0.5 g/l;
Tryptone, 20 g/l; KCI, 2.5 mM; MgCl
2, 10 mM) containing ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose is used
for inducing the plasmid containing the genes of the Red system) and grown at 30 °C
to reach the optical density of the bacterial culture OD
600=0.4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
with ice-cold de-ionized water, followed by suspension in 100 µl of the water. 10
µl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells
were added to 1-ml of SOC medium (
Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring
Harbor Laboratory Press (1989)), incubated for 2 hours at 37°C, and then were spread onto L-agar containing 25
µg/ml of chloramphenicol. Colonies grown for 24 hours were tested for the presence
of Cm
R marker instead of the native
tdh gene by PCR using primers P3 (SEQ ID NO: 5) and P4 (SEQ ID NO: 6). For this purpose,
a freshly isolated colony was suspended in 20µl water and then 1µl of obtained suspension
was used for PCR. The temperature profile was the following: initial DNA denaturation
for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec, annealing
at 55 °C for 30 sec and elongation at 72 °C for 30 sec; the final elongation for 5
min at 72 °C. A few Cm
R colonies tested contained the desired 1104 bp DNA fragment, confirming the presence
of Cm
R marker DNA instead of 1242 bp fragment of
tdh gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46
by culturing at 37 °C and the resulting strain was named
E.
coli MG1655Δtdh.
[0112] Then, the rhtA23 mutation from the strain VL614rhtA23 (
Livshits V.A. et al, 2003, Res. Microbiol., 154:123-135) was introduced into the obtained strain MG1655 Δtdh resulting in strain MG1655 Δtdh,
rhtA*. The rhtA23 is a mutation which confers resistance to high concentrations of
threonine (>40 mg/ml) and homoserine (>5 mg/ml). For that purpose the strain MG1655
Δtdh was infected with phage P1
vir grown on the donor strain VL614rhtA23. The transductants were selected on M9 minimal
medium containing 8 mg/ml homoserine and 0.4% glucose as the sole carbon source.
Example 2. Construction of E. coli MG1655::PL-tacadhE
[0113] E. coli MG1655::P
L-tacadh was obtained by replacement of the native promoter region of the
adhE gene in the strain MG1655 by P
L-tac promoter.
[0114] To replace the native promoter region of the
adhE gene, the DNA fragment carrying a P
L-tac promoter and chloramphenicol resistance marker (Cm
R) encoded by the
cat gene was integrated into the chromosome of
E.
coli MG1655 in the place of the native promoter region by the method described by
Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645), which is also called "Red-mediated integration" and/or "Red-driven integration".
[0115] A fragment containing the P
L-tac promoter and the
cat gene was obtained by PCR using chromosomal DNA of
E. coli MG1655P
L-tacxylE (
WO2006/043730) as a template. The nucleotide sequence of the P
L-tac promoter is presented in the Sequence listing (SEQ ID NO: 7). Primers P5 (SEQ ID
NO: 8) and P6 (SEQ ID NO: 9) were used for PCR amplification. Primer P5 contains 40
nucleotides complementary to the region located 318 bp upstream of the start codon
of the
adhE gene introduced into the primer for further integration into the bacterial chromosome
and primer P6 contains a 39 nucleotides identical to 5'-sequence of the
adhE gene.
[0116] PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x PCR-buffer with
15 mM MgCl
2 ("Fermentas", Lithuania), 200 µM each of dNTP, 25 pmol each of the exploited primers
and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 20 ng of the
E. coli MG1655P
L-tacxylE genomic DNA was added in the reaction mixtures as a template for PCR.
[0117] The temperature profile was the following: initial DNA denaturation for 5 min at
95 °C, followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 54
°C for 30 sec, elongation at 72 °C for 1.5 min and the final elongation for 5 min
at 72 °C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis,
extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.
The obtained DNA fragment was used for electroporation and Red-mediated integration
into the bacterial chromosome of the
E. coli MG1655/pKD46.
[0118] MG1655/pKD46 cells were grown overnight at 30 °C in the liquid LB-medium containing
ampicillin (100 µg/ml), then diluted 1:100 by SOB-medium (Yeast. extract, 5 g/l; NaCl,
0.5 g/l; Tryptone, 20 g/l; KCI, 2.5 mM; MgCl
2, 10 mM) containing ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose is used
for inducing the plasmid encoding genes of the Red system) and grown at 30 °C to reach
the optical density of the bacterial culture OD
600=0,4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
with ice-cold de-ionized water, followed by suspension in 100 µl of the water. 10
µl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions.
[0120] About 100 resulting clones were selected on M9 plates with 2% ethanol as the sole
carbon source. Some clones which grew on M9 plates with 2% ethanol in 36 hours were
chosen and tested for the presence of Cm
R marker instead of the native promoter region of the
adhE gene by PCR using primers P7 (SEQ ID NO: 10) and P8 (SEQ ID NO: 11). For this purpose,
a freshly isolated colony was suspended in 20µl water and then 1µl of the obtained
suspension was used for PCR. The temperature profile follows: initial DNA denaturation
for 10 min at 95 °C; then 30 cycles of denaturation at 95°C for 30 sec, annealing
at 54 °C for 30 sec and elongation at 72°C for 1.5min; the final elongation for 1
min at 72°C. A few Cm
R colonies tested contained the desired ∼1800 bp DNA fragment, confirming the presence
of Cm
R marker DNA instead of 520 bp native promoter region of
adhE gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46
by culturing at 37 °C and the resulting strain was named
E. coli MG1655::P
L-tacadhE (See Figure 1).
Example 3. Construction of E. coli MG1655Δtdh, rhtA*, PL-tacadhE
[0121] E. coli MG1655Δtdh, rhtA*, P
L-tacadhE was obtained by transduction of the P
L-tac promoter from the strain MG1655::P
L-tacadhE into strain MG1655Δtdh, rhtA*.
[0122] The strain MG1655Δtdh, rhtA* was infected with phage Pl
vir grown on the donor strain MG1655::P
L-tacadhE, and the strain MG1655Δtdh, rhtA*, P
L-tacadhE was obtained. This strain was checked for growth on M9 plates with 2% ethanol
as the sole carbon source. The growth rate was the same as for the strain MG1655::P
L-tacadhE.
Example 4. The effect of increasing the adhE gene expression on L-threonine production
To evaluate the effect of enhancing expression of the adhE gene on L-threonine production, both E. coli strains MG1655Δtdh, rhtA*, PL-tacadhE and MG1655Δtdh, rhtA* were transformed with plasmid pVIC40.
[0123] The strain MG1655Δtdh, rhtA*, P
L-tacadhE (pVIC40) and a parent strain MG1655Δtdh, rhtA* (pVIC40) were each cultivated
at 37 °C for 18 hours in a nutrient broth and 0.3 ml of each of the obtained cultures
was inoculated into 3 ml of fermentation medium having the following composition in
a 20x200 mm test tube and cultivated at 34°C for 48 hours with a rotary shaker. Data
from at least 10 independent experiments are shown on Tables 1 and 2.
[0124] Fermentation medium composition (g/l):
Ethanol |
24 or 16 |
Glucose |
0 (Table 1) or 3 (Table 2) |
(NH4)2SO4 |
16 |
K2HPO4 |
0.7 |
MgSO4·7H2O |
1.0 |
MnSO4·5H2O |
0.01 |
FeSO4·7H2O |
0.01 |
Thiamine hydrochloride |
0.002 |
Yeast extract |
1.0 |
L-isoleucine |
0.01 |
CaCO3 |
33 |
MgSO4·7H2O and CaCO3 were each sterilized separately. |
[0125] It can be seen from the Tables 1 and 2, MG1655Δtdh, rhtA*, P
L-tacadhE was able to accumulate a higher amount of L-threonine as compared with MG1655Δtdh,
rhtA*. Moreover, MG1655Δtdh, rhtA*, P
L-tacadhE was able to grow on the medium containing ethanol as the sole carbon source and
cause accumulation of L-threonine, whereas MG1655Δtdh, rhtA* exhibited very poor growth
and productivity in the medium containing ethanol as the sole carbon source.
Example 5. Construction of E. coli MG1655ΔadhE
[0126] This strain was constructed by inactivation of the native
adhE gene in
E.
coli MG1655 by the
kan gene.
[0127] To inactivate (or disrupt) the native
adhE gene, the DNA fragment carrying kanamycin resistance marker (Km
R) encoded by the
kan gene was integrated into the chromosome of
E.
coli MG1655 (ATCC 700926) in place of the native gene by the method described by
Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97,6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration".
[0128] A DNA fragment containing a Km
R marker (kan gene) was obtained by PCR using the commercially available plasmid pACYC177
(GenBank/EMBL accession number X06402, "Fermentas", Lithuania) as the template, and
primers P9 (SEQ ID NO: 12) and P10 (SEQ ID NO: 13). Primer P9 contains 40 nucleotides
homologous to the region located 318 bp upstream of the start codon of the
adhE gene introduced into the primer for further integration into the bacterial chromosome.
Primer P10 contains 41 nucleotides homologous to the 3'-region of the
adhE gene introduced into the primer for further integration into the bacterial chromosome.
[0129] PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x PCR-buffer with
25 mM MgCl
2 ("Fermentas", Lithuania), 200 µM each of dNTP, 25 pmol each of the exploited primers
and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 5 ng of the plasmid
DNA was added in the reaction mixture as a template DNA for the PCR amplification.
The temperature profile was the following: initial DNA denaturation for 5 min at 95
°C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C
for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at
72 °C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis,
extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.
[0130] The obtained DNA fragment was used for electroporation and Red-mediated integration
into the bacterial chromosome of the
E. coli MG1655/pKD46.
[0131] MG1655/pKD46 cells were grown overnight at 30 °C in liquid LB-medium containing ampicillin
(100 µg/ml), then diluted 1:100 by SOB-medium (Yeast extract, 5 g/l; NaCl, 0.5 g/l;
Tryptone, 20 g/l; KCI, 2.5 mM; MgCl
2, 10 mM) containing ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose is used
for inducing the plasmid encoding genes of Red system) and grown at 30 °C to reach
the optical density of the bacterial culture OD
600=0,4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
by the ice-cold de-ionized water, followed by suspension in 100 µl of the water. 10
µl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells
were added to 1-ml of SOC medium (
Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring
Harbor Laboratory Press (1989)), incubated for 2 hours at 37°C, and then were spread onto L-agar containing 20
µg/ml of kanamycin. Colonies grown within 24 hours were tested for the presence of
Km
R marker instead of the native
adhE gene by PCR using primers P11 (SEQ ID NO: 14) and P12 (SEQ ID NO: 15). For this purpose,
a freshly isolated colony was suspended in 20µl water and then 1µl of obtained suspension
was used for PCR. The temperature profile follows: initial DNA denaturation for 5
min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec, annealing at 55
°C for 30 sec and elongation at 72 °C for 30 sec; the final elongation for 5 min at
72 °C. A few Km
R colonies tested contained the desired about 1030 bp DNA fragment, confirming the
presence of Km
R marker DNA instead of the 3135 bp fragment of
adhE gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46
by culturing at 37 °C and the resulting strain was named
E.
coli MG1655ΔadhE.
Example 6. Construction of E. coli MG1655::PL-tacadhE*
[0132] E. coli MG1655::P
L-tacadhE* was obtained by introduction of the Glu568Lys (E568K) mutation into the
adhE gene. First, 1.05 kbp fragment of the
adhE gene carrying the E568K mutation was obtained by PCR using the genomic DNA of
E.
coli MG1655 as the template and primers P13 (SEQ ID NO: 16) and P12 (SEQ ID NO: 15). Primer
P15 homologous to 1662-1701 bp and 1703-1730 bp regions of the
adhE gene and includes the substitution
g/a (position 1702 bp) shown as bold and primer P12 homologous to 3'-end of the
adhE gene. PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied
Biosystems). The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x
PCR-buffer with MgCl
2 ("TaKaRa", Japan), 250 µM each of dNTP, 25 pmol each of the exploited primers and
2.5 U of Pyrobest DNA polymerase ("TaKaRa", Japan). Approximately 20 ng of the
E. coli MG1655 genomic DNA was added in the reaction mixtures as a template for PCR. The
temperature profile was the following: initial DNA denaturation for 5 min at 95 °C,
followed by 35 cycles of denaturation at 95 °C for 30 sec, annealing at 54 °C for
30 sec, elongation at 72 °C for 1min and the final elongation for 5 min at 72 °C.
The fragment obtained was purified by agarose gel-electrophoresis, extracted using
"GenElute Spin Columns" ("Sigma", USA) and precipitated with ethanol.
[0133] In the second step, the fragment containing the P
L-tac promoter with the mutant
adhE gene and marked by the
cat gene, which provides chloramphenicol resistance, was obtained by PCR using the genomic
DNA of
E. coli MG1655::P
L-tacadhE as the template (see Example 2), primer P11 (SEQ ID NO: 14) and a 1.05 kbp fragment
carrying a mutant sequence (see above) as a second primer. Primer P11 is homologous
to the region located at 402-425 bp upstream of the start codon of the
adhE gene. PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied
Biosystems). The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x
PCR-buffer ("TaKaRa", Japan), 25mM MgCl
2, 250 µM each of dNTP, 10 ng of the primer P11, 1µg of the 1.05 kbp fragment as a
second primer and 2.5U of TaKaRa LA DNA polymerase ("TaKaRa", Japan). Approximately
20 ng of the
E. coli MG1655::P
L-tacadhE genomic DNA was added to the reaction mixture as a template for PCR. The temperature
profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by
35 cycles of denaturation at 95 °C for 30 sec, annealing at 54 °C for 30 sec, elongation
at 72 °C for 3.5 min and the final elongation for 7 min at 72 °C. The resulting fragment
was purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns"
("Sigma", USA) and precipitated by ethanol.
[0134] To replace the native region of the
adhE gene, the DNA fragment carrying a P
L-tac promoter with the mutant
adhE and chloramphenicol resistance marker (Cm
R) encoded by the
cat gene (cat-P
L-tacadhE*, 4.7 kbp) was integrated into the chromosome of
E.
coli MG1655ΔadhE by the method described by
Datsenko K.A. and Wanner B.L. (Proc. Natl. Acad. Sci. USA, 2000, 97, 6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration".
MG1655 ΔadhE/pKD46 cells were grown overnight at 30 °C in liquid LB-medium containing
ampicillin (100 µg/ml), then diluted 1:100 by SOB-medium (Yeast extract, 5 g/l; NaCl,
0.5 g/l; Tryptone, 20 g/l; KCI, 2.5 mM; MgCl
2, 10 mM) containing ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose is used
for inducing the plasmid encoding genes of the Red system) and grown at 30 °C to reach
the optical density of the bacterial culture OD
600=0,4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
by the ice-cold de-ionized water, followed by suspension in 100 µl of the water. 10
µl of DNA fragment (300 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions.
[0136] The clones obtained were selected on M9 plates with 2% ethanol as the sole carbon
source.
[0137] The runaway clone was chosen and the full gene sequence was verified. The row of
mutations was revealed as follows: Glu568Lys (gag - aag), Ile554Ser (atc - agc), Glu22Gly
(gaa -gga), Met236Val (atg - gtg), Tyr461 Cys (tac - tgc), Ala786Val (gca - gta).
This clone was named MG1655::P
L-tacadhE*.
Example 7. Construction of E. coli MG1655Δtdh, rhtA*, PL-tacadhE*
[0138] E. coli MG1655Δtdh, rhtA*, P
L-tacadhE* was obtained by transduction of the P
L-tac adhE* mutation from the strain MG1655::P
L-tacadhE*.
[0139] The strain MG1655Δtdh, rhtA* was infected with phage P1
vir grown on the donor strain MG1655::P
L-tacadhE* and the strain MG1655Δtdh, rhtA*, P
L-tacadhE* was obtained. This strain was checked for growth on M9 plates with 2% ethanol
as a sole carbon source. The growth rate was the same as for the strain MG1655::P
L-tacadhE*.
Example 8. Construction of E. coli MG1655Δtdh, rhtA*, PL-tacadhE-Lys568
[0140] A second attempt to obtain a single mutant
adhE having the Glu568Lys mutation was performed. For that purpose
E.
coli strain MG1655Δtdh, rhtA*, P
L-tacadhE-wtΔ34 was constructed.
[0141] E. coli MG1655Δtdh, rhtA*, P
L-tacadhE-wtΔ34 was obtained by replacement of a 34bp fragment of the
adhE gene (the region from 1668 to 1702 bp, inclusive of the triplet encoding Glu568)
in
E.
coli MG1655Δtdh, rhtA*, P
L-tacadhE-wt (wt means a wild type) with
kan gene. The
kan gene was integrated into the chromosome of
E.
coli MG1655Δtdh, rhtA*, P
L-
tacadhE-wt by the method, described by
Datsenko K.A. and Wanner B.L. (Proc.Natl.Acad.Sci.USA, 2000, 97, 6640-6645) which is also called "Red-mediated integration" and/or "Red-driven integration".
[0142] A DNA fragment containing a Km
R marker encoded by the
kan gene was obtained by PCR using the commercially available plasmid pACYC177 (GenBank/EMBL
accession number X06402, "Fermentas", Lithuania) as the template, and primers P 14
(SEQ ID NO: 17) and P15 (SEQ ID NO: 18). Primer P14 contains 41 nucleotides identical
to the region from 1627 to 1668 bp of
adhE gene and primer P 15 contains 39 nucleotides complementary to the region from 1702
to 1740 bp of
adhE gene introduced into the primers for further integration into the bacterial chromosome.
[0143] PCR was provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x PCR-buffer with
25 mM MgCl
2 ("Fermentas", Lithuania), 200 µM each of dNTP, 25 pmol each of the exploited primers
and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 5 ng of the plasmid
DNA was added in the reaction mixture as a template DNA for the PCR amplification.
The temperature profile was the following: initial DNA denaturation for 5 min at 95
°C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C
for 30 sec, elongation at 72 °C 50 sec and the final elongation for 5 min at 72 °C.
Then, the amplified DNA fragment was purified by agarose gel-electrophoresis, extracted
using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.
[0144] Colonies obtained were tested for the presence of Km
R marker by PCR using primers P16 (SEQ ID NO: 19) and P17 (SEQ ID NO: 20). For this
purpose, a freshly isolated colony was suspended in 20µl water and then 1µl of the
obtained suspension was used for PCR. The temperature profile follows: initial DNA
denaturation for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec,
annealing at 55 °C for 30 sec and elongation at 72° C for 45 sec; the final elongation
for 5 min at 72 °C. A few Km
R colonies tested contained the desired 1200 bp DNA fragment, confirming the presence
of Km
R marker DNA instead of 230 bp fragment of native
adhE gene. One of the obtained strains was cured of the thermosensitive plasmid pKD46
by culturing at 37 °C and the resulting strain was named as
E. coli MG1655Δtdh, rhtA*,P
L-tacadhE-wtΔ34.
[0145] Then, to replace the kanamycin resistance marker (Km
R) encoded by
kan gene with a fragment of the
adhE gene encoding the Glu568Lys mutation, the oligonucleotides P18 (SEQ ID NO: 21) and
P19 (SEQ ID NO: 22) carrying the appropriate mutation were integrated into the chromosome
of
E.
coli MG1655Δtdh, rhtA, P
L-tacadhE-wt Δ34 by the method "Red-mediated integration" and/or "Red-driven integration"
(
Yu D., Sawitzke J. et al., Recombineering with overlapping single-stranded DNA oligonucleotides:
Testing of recombination intermediate, PNAS, 2003, 100(12), 7207-7212). Primer P18 contains 75 nucleotides identical to the region from 1627 to 1702 bp
of
adhE gene and primer P 19 contains 75 nucleotides complementary to the region from 1668
to 1740 bp of
adhE gene, both primers inclusive of the triplet encoding Lys568 instead of Glu568.
[0146] The clones were selected on M9 minimal medium containing 2% ethanol and 25mg/ml succinate
as a carbon source.
[0147] Colonies were tested for the absence of Km
R marker by PCR using primers P16 (SEQ ID NO: 19) and P17 (SEQ ID NO: 20). For this
purpose, a freshly isolated colony was suspended in 20µl water and then 1µl of the
obtained suspension was used for PCR. The temperature profile follows: initial DNA
denaturation for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec,
annealing at 55 °C for 30 sec and elongation at 72°C for 25 sec; the final elongation
for 5 min at 72 °C. A few Km
S colonies tested contained the desired 230 bp DNA fragment of
adhE gene, confirming the absence of Km
R marker DNA instead of 1200 bp fragment. Several of the obtained strains was cured
of the thermosensitive plasmid pKD46 by culturing at 37 °C and the resulting strain
was named as
E. coli MG1655Δtdh, rhtA, P
L-tacadhE-Lys568.
[0148] The presence of the Glu568Lys mutation was confirmed by sequencing, for example,
cl.18 has a single mutation Glu568Lys. Addditionally it was found that some clones
(#1, 13) contained additional mutations: cl. 1 - Glu568Lys, Phe566Val; cl.13 - Glu568Lys,
Glu560Lys.
[0149] For strains MG1655Δtdh, rhtA*,P
L-tacadhE-Lys568 (cl.18), MG1655Δtdh, rhtA*,P
L-
tacadhE-Lys568,Val566 (cl.1), MG1655Δtdh, rhtA*,P
L-tacadhE-Lys568,Lys560 (cl.13) and MG1655Δtdh, rhtA*,P
L-tacadhE*, the growth curves were studied (Figures 3 and 4).
[0150] The strains were grown in M9 medium with ethanol as a sole carbon source and in M9
medium with glucose and ethanol (molar ratio 1:3)
Example 9. Construction of E. coli MG1655Δtdh, rhtA*, adhE*
[0151] The
E. coli strain MG1655Δtdh, rhtA*, adhE* was obtained by reconstruction of the native
adhE promoter in strain MG1655Δtdh, rhtA*, P
L-tacadhE*. A DNA fragment carrying a P
L-tac promoter and chloramphenicol resistance marker (Cm
R) encoded by
cat gene in the chromosome of the strain MG1655Δtdh, rhtA*, P
L-tacadhE* was replaced by a fragment carrying native
adhE promoter and kanamycin resistance marker (Km
R) encoded by the
kan gene. Native P
adhE was obtained by PCR using a DNA of the strain MG1655 as a template and primers P20
(SEQ ID NO: 23) and P21 (SEQ ID NO: 24). Primer P20 contains an
EcoRI recognition site at the 5'-end thereof, which is necessary for further joining to
the
kan gene and primer P21 contains 30 nucleotides homologous to 5'-region of the adhE gene
(from 50 bp to 20 bp).
[0152] A DNA fragment containing a Km
R marker encoded by the
kan gene was obtained by PCR using the commercially available plasmid pACYC177 (GenBank/EMBL
accession number X06402, "Fermentas", Lithuania) as the template, and primers P22
(SEQ ID NO: 25) and P23 (SEQ ID NO: 26). Primer P22 contains 41 nucleotides homologous
to the region located 425 bp upstream of the start codon of the
adhE gene introduced into the primer for further integration into the bacterial chromosome
and primer P23 contains an
EcoRI recognition site at the 3'-end thereof, which is necessary for further joining
to the P
adhE promoter.
[0153] PCR were provided using the "Gene Amp PCR System 2700" amplificatory (Applied Biosystems).
The reaction mixture (total volume - 50 µl) consisted of 5 µl of 10x PCR-buffer with
25 mM MgCl
2 ("Fermentas", Lithuania), 200 µM each of dNTP, 25 pmol each of the exploited primers
and 1 U of Taq-polymerase ("Fermentas", Lithuania). Approximately 20 ng of genomic
DNA or 5 ng of the plasmid DNA were added in the reaction mixture as a template for
the PCR amplification. The temperature profile was the following: initial DNA denaturation
for 5 min at 95 °C, followed by 35 cycles of denaturation for P
adhE or 25 cycles of denaturation for
kan gene at 95 °C for 30 sec, annealing at 55 °C for 30 sec, elongation at 72 °C for
20 sec for Ptac promoter and 50 sec for
kan gene; and the final elongation for 5 min at 72 °C. Then, the amplified DNA fragments
were purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns"
("Sigma", USA) and precipitated by ethanol.
[0154] Each of the two above-described DNA fragments was treated with
EcoRI restrictase and ligated. The ligation product was amplified by PCR using primers
P21 and P22. The amplified
kan-P
adhE DNA fragment was purified by agarose gel-electrophoresis, extracted using "GenElute
Spin Columns" ("Sigma", USA) and precipitated by ethanol. The obtained DNA fragment
was used for electroporation and Red-mediated integration into the bacterial chromosome
of the
E. coli MG1655Δtdh::rhtA*, P
L-tacadhE*/pKD46.
[0155] MG1655Atdh::rhtA*,P
L-tacdhE*/pKD46 cells were grown overnight at 30 °C in the liquid LB-medium with addition
of ampicillin (100 µg/ml), then diluted 1:100 by the SOB-medium (Yeast extract, 5
g/l; NaCl, 0.5 g/l; Tryptone, 20 g/l; KCl, 2.5 mM; MgCl
2, 10 mM) with addition of ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose
was used for inducing the plasmid encoding genes of Red system) and grown at 30 °C
to reach the optical density of the bacterial culture OD
600=0,4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
by the ice-cold de-ionized water, followed by suspending in 100 µl of the water. 10
µl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions.
[0157] Colonies grown within 24 h were tested for the presence of P
adhE -Km
R marker instead of P
L-tac-Cm
R-marker by PCR using primers P24 (SEQ ID NO: 27) and P25 (SEQ ID NO: 28). For this
purpose, a freshly isolated colony was suspended in 20µl water and then 1 µl of obtained
suspension was used for PCR. The temperature profile follows: initial DNA denaturation
for 5 min at 95 °C; then 30 cycles of denaturation at 95 °C for 30 sec, annealing
at 54 °C for 30 sec and elongation at 72 °C for 1.0 min; the final elongation for
5 min at 72 °C. A few Km
R colonies tested contained the desired 1200 bp DNA fragment, confirming the presence
of native P
adhE promoter and Km
R -marker DNA. Some of these fragments were sequenced. The structure of the native
P
adhE promoter was confirmed. One of the strains containing the mutant
adhE gene under the control of anative promoter was cured of the thermosensitive plasmid
pKD46 by culturing at 37 °C and the resulting strain was named as
E.
coli MG1655Δtdh, rhtA*, adhE*.
[0158] The ability of all the obtained strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-
tacadhE-Lys568, Val566 (cl.1); MG1655Δtdh, rhtA*, adhE* and parental strain MG1655Δtdh,
rhtA* to grow on the minimal medium M9 containing ethanol as a sole carbon source
was investigated. It was shown that the parental strain MG1655Δtdh, rhtA* and the
strain with enhanced expression of wild-type alcohol dehydrogenase were unable to
grow on the medium containing ethanol (2% or 3%) as a sole carbon source (Figure 3,
A and B). Strain MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18) containing the single mutation in the alcohol dehydrogenase described
early (
Membrillo-Hernandez, J. et al, J. Biol. Chem. 275, 33869-33875 (2000)) exhibited very poor growth in the same medium. But strains containing mutations
in the alcohol dehydrogenase in addition to mutation Glu568Lys exhibited good growth
(Figure 3, A and B). All the above strains were able to grow on the minimal medium
M9 containing a mixture of glucose and ethanol, but strains with enhanced expression
of the mutant alcohol dehydrogenase containing mutations in addition to mutation Glu568Lys
exhibited better growth (Figure 4).
[0159] It was also shown that strain MG1655Δtdh, rhtA*, adhE* containing the alcohol dehydrogenase
with 5 mutations under the control of the native promoter was unable to grow on the
minimal medium M9 containing ethanol (2% or 3%) as a sole carbon source. Enhanced
expression of the gene encoding for said alcohol dehydrogenase is necessary for good
growth (Figure 5).
Example 10. The effect of increasing the mutant adhE gene expression on L-threonine production
[0160] To evaluate the effect of enhancing expression of the mutant
adhE gene on threonine production,
E. coli strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566 (cl.1); MG1655Δtdh, rhtA*, adhE* and parental strain MG1655Δtdh,
rhtA* were transformed with plasmid pVIC40.
[0161] These strains and the parent strain MG1655Δtdh, rhtA* (pVIC40) were cultivated at
37 °C for 18 hours in a nutrient broth and 0.3 ml of each of the obtained cultures
was inoculated into 3 ml of fermentation medium (see Example 4) in a 20x200 mm test
tube and cultivated at 34 °C for 48 hours with a rotary shaker. Data from at least
10 independent experiments are shown on Tables 1 and 2.
[0162] It can be seen from the Tables 1 and 2, mutant alcohol dehydrogenase was able to
cause accumulation of a higher amount of L-threonine as compared with MG1655Δtdh,
rhtA* in which neither expression of a wild-type nor a mutant alcohol dehydrogenase
was increased or even with MG1655Δtdh, rhtA*, or P
L-tacadhE, in which expression of wild-type alcohol dehydrogenase was increased. Such higher
accumulation of L-threonine during fermentation was observed in the medium containing
either a mixture of glucose and ethanol, or just ethanol as the sole carbon source.
Table 1
Strain |
3% ethanol |
2% ethanol |
OD540 |
Thr, g/l |
OD540 |
Thr, g/l |
MG1655Δtdh,rhtA* (pVIC40) |
1.6±0.1 |
<0.1 |
1.4±0.1 |
<0.1 |
MG1655Δtdh, rhtA*,PL-tacadhE(wt) (pVIC40) |
7.9±0.3 |
1.1±0.1 |
7.6±0.2 |
0.9±0.1 |
MG1655Δtdh, rhtA*, PL-tacadhE-Lys568(pVIC40)(cl.18) |
14.7±0.3 |
3.3±0.1 |
13.7±0.4 |
2.3±0.3 |
MG1655Δtdh, rhtA*, PL-tacadhE-Lys568, Val566(pVIC40) (cl.1) |
14.2±0.4 |
3.2±0.2 |
12.5±0.3 |
2.1±0.3 |
MG1655Δtdh, rhtA*, PL-tacadhE*(pVIC40) |
17.0±0.3 |
3.9±0.2 |
14.3±0.3 |
2.8±0.1 |
MG1655Δtdh, rhtA*, adhE*(pVIC40) |
2.8±0.2 |
<0.1 |
2.1±0.1 |
< 0.1 |
Table 2
Strain |
2,7% ethanol+0,3% glucose |
OD540 |
Thr, g/l |
MG1655Δtdh, rhtA* (pVIC40) |
6.6±0.2 |
0.9±0.2 |
MG1655Δtdh, rhtA*,PL-tacadhE(wt) (pVIC40) |
13.4±0.3 |
1.4±0.3 |
MG1655Δtdh, rhtA*, PL-tacadhE-Lys568 (pVIC40) (cl.18) |
16.1±0.4 |
2.6±0.2 |
MG1655Δtdh, rhtA*, PL-tacadhE-Lys568, Val566(pVIC40) (cl.1) |
15.5±0.3 |
2.9 ±0.2 |
MG1655Δtdh, rhtA*, PL-tacadhE*(pVIC40) |
18.8±0.4 |
2.8±0.1 |
MG1655Δtdh, rhtA*, adhE*(pVIC40) |
5.8±0.1 |
0.8±0.3 |
[0163] Test-tube fermentation was carried out without reversion of evaporated ethanol.
Example 11. The effect of increasing adhE gene expression on L-lysine production
[0164] To test the effect of enhanced expression of the
adhE gene under the control of P
L-tac promoter on lysine production, the DNA fragments from the chromosome of the above-described
strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566 (cl.1); MG1655Δtdh, rhtA*, adhE* were transferred to the lysine-producing
E.
coli strain WC1960ΔcadAΔldc (pCABD2) by P1 transduction (
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,
Plainview, NY). pCABD2 is a plasmid comprising the
dapA gene coding for a dihydrodipicolinate synthase having a mutation which desensitizes
feedback inhibition by L-lysine, the
lysC gene coding for aspartokinase III having a mutation which desensitizes feedback inhibition
by L-lysine, the
dapB gene coding for a dihydrodipicolinate reductase, and the
ddh gene coding for diaminopimelate dehydrogenase (
U.S. Patent No. 6,040,160).
[0165] The resulting strains and the parent strain WC196ΔcadAΔldc (pCABD2) were spread on
L-medium plates containing 20 mg/l of streptomycin at 37 °C, and cells corresponding
to 1/8 of a plate were inoculated into 20 ml of the fermentation medium containing
the required drugs in a 500 ml-flask. The cultivation can be carried out at 37 °C
for 48 hours by using a reciprocal shaker at the agitation speed of 115 rpm. After
the cultivation, the amounts of L-lysine and residual ethanol in the medium can be
measured by a known method (Bio-Sensor BF-5, manufactured by Oji Scientific Instruments).
Then, the yield of L-lysine relative to consumed ethanol can be calculated for each
of the strains.
[0166] The composition of the fermentation medium (g/l) was as follows:
Ethanol |
20.0 |
(NH4)2SO4 |
24.0 |
K2HPO4 |
1.0 |
MgSO4·7H2O |
1.0 |
FeSO4·7H2O |
0.01 |
MnSO4·5H2O |
0.01 |
Yeast extract |
2.0 |
[0167] pH is adjusted to 7.0 by KOH and the medium was autoclaved at 115°C for 10 min. Ethanol
and MgSO
4 7H
2O were sterilized separately. CaCO
3 was dry-heat sterilized at 180°C for 2 hours and added to the medium at a final concentration
of 30 g/l. Data from two parallel experiments are shown on Table 3.
Table 3
Strain |
2% ethanol |
OD600 |
Lys, g/l |
WC196ΔcadAΔldc (pCABD2) |
1.1±0.0 |
0.2±0.0 |
WC196ΔcadAΔldc,PL-tacadhE(wt) (pCABD2) |
1.5±0.1 |
0.4±0.1 |
MG1655ΔcadAΔldc, PL-tacadhE-Lys568 (pCABD2) (cl.18) |
5.2±0.4 |
1.3±0.1 |
MG1655ΔcadAΔldc, PL-tacadhE-Lys568, Val566(pCABD2) (cl.1) |
1.6±0.0 |
0.8 ±0.3 |
MG1655ΔcadAΔldc, PL-tacadhE*(pCABD2) |
5.9±0.2 |
1.8±0.1 |
[0168] It can be seen from Table 3 that mutant alcohol dehydrogenases and a wild-type alcohol
dehydrogenase was able to cause growth enhancement and accumulation of a higher amount
of L-lysine as compared with WC 196ΔcadAΔldc (pCABD2), in which neither expression
of a wild-type nor a mutant alcohol dehydrogenase was increased.
Example 12. Construction of E. coli MG1655ΔargR,PL-tacadhE*
1. Construction of the strain MG1655ΔargR
[0169] This strain was constructed by inactivation of the native
argR gene, which encodes a repressor of the L-arginine biosynthetic pathway in
E. coli MG1655 by the
kan gene. To replace the native
argR gene, the DNA fragment carrying a kanamycin resistance marker (Km
R) encoded by the
kan gene was integrated into the chromosome of
E.
coli MG1655 (ATCC 700926) in place of the native
argR gene by the Red-driven integration.
[0170] A DNA fragment containing a Km
R marker encoded by the
kan gene was obtained by PCR using the commercially available plasmid pACYC177 (GenBank/EMBL
accession number X06402, "Fermentas", Lithuania) as a template, and primers P26 (SEQ
ID NO: 31) and P27 (SEQ ID NO: 32). Primer P26 contains 40 nucleotides homologous
to the 5'-region of the
argR gene introduced into the primer for further integration into the bacterial chromosome.
Primer P27 contains 41 nucleotides homologous to the 3'-region of the
argR gene introduced into the primer for further integration into the bacterial chromosome.
The temperature profile was the following: initial DNA denaturation for 5 min at 95
°C, followed by 25 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C
for 30 sec, elongation at 72 °C for 40 sec; and the final elongation for 5 min at
72 °C. Then, the amplified DNA fragment was purified by agarose gel-electrophoresis,
extracted using "GenElute Spin Columns" ("Sigma", USA) and precipitated by ethanol.
[0171] The obtained DNA fragment was used for electroporation and Red-mediated integration
into the bacterial chromosome of the
E.
coli MG1655/pKD46.
[0172] MG1655/pKD46 cells were grown overnight at 30 °C in the liquid LB-medium with addition
of ampicillin (100µg/ml), then diluted 1:100 by the SOB-medium (Yeast extract, 5 g/l;
NaCl, 0.5 g/l; Tryptone, 20 g/l; KCI, 2.5 mM; MgCl
2, 10 mM) with the addition of ampicillin (100 µg/ml) and L-arabinose (10 mM) (arabinose
is used for inducing the plasmid encoding genes of Red system) and grown at 30 °C
to reach the optical density of the bacterial culture OD
600=0,4-0.7. The grown cells from 10 ml of the bacterial culture were washed 3 times
by the ice-cold de-ionized water, followed by suspending in 100 µl of the water. 10
µl of DNA fragment (100 ng) dissolved in the de-ionized water was added to the cell
suspension. The electroporation was performed by "Bio-Rad" electroporator (USA) (No.
165-2098, version 2-89) according to the manufacturer's instructions. Shocked cells
were added to 1-ml of SOC medium, incubated 2 hours at 37°C, and then were spread
onto L-agar containing 25 µg/ml of chloramphenicol. Colonies grown within 24 h were
tested for the presence of Km
R marker instead of the native
argR gene by PCR using primers P28 (SEQ ID NO: 33) and P29 (SEQ ID NO: 34). For this purpose,
a freshly isolated colony was suspended in 20µl water and then 1µl of obtained suspension
was used for PCR. The temperature profile was the following: initial DNA denaturation
for 5 min at 95 °C; then 30 cycles of denaturation at 95 °c for 30 sec, annealing
at 55 °C for 30 sec and elongation at 72 °C for 30 sec; the final elongation for 5
min at 72 °C. A few Km
R colonies tested contained the desired 1110 bp DNA fragment, confirming the presence
of Km
R marker DNA instead of 660 bp fragment of
argR gene. One of the obtained strains was cured from the thermosensitive plasmid pKD46
by culturing at 37 °C and the resulting strain was named
E. coli MG1655ΔargR.
2. Construction of E. coli MG1655ΔargR,PL-tacadhE*.
[0173] E. coli MG16550argR,P
L-tacadhE* was obtained by transduction of the P
L-tac adhE* mutation from the strain MG1655::P
L-tacadhE*.
[0174] The strain MG1655ΔargR was infected with phage P1
vir grown on the donor strain MG1655::P
L-tacadhE* and the strain MG1655ΔargR,P
L-tacadhE* was obtained. This strain was checked for growth on M9 plates with 2% ethanol
as a sole carbon source. The growth rate was the same as for the strain MG1655::P
L-tacadhE* (36 h).
Example 13. Construction of the pMW119-ArgA4 plasmid
[0175] ArgA gene with a single mutation provide the fbr (feedback resistant) phenotype (
JP2002253268,
EP1170361) and under the control of its own promoter was cloned into pMW119 vector.
[0176] The
argA gene was obtained by PCR using the plasmid pKKArgA-r4 (
JP2002253268,
EP1170361) as a template, and primers P30 (SEQ ID NO: 35) and P31 (SEQ ID NO: 36). Sequence
of the primer P30 homologous to the 5'-region of the
argA gene located 20 bp upstream and 19 bp downstream of the start codon of the
argA gene. Primer P31 contains 24 nucleotides homologous to the 3'-region of
argA gene and
HindIII restriction site introduced for further cloning into the pMW 119/
BamHI-HindIII vector.
[0177] Sequence of the P
argA promoter was obtained by PCR using
E. coli MG1655 as a template, and primers P32 (SEQ ID NO: 37) and P33 (SEQ ID NO: 38). Primer
P32 contains 30 nucleotides homologous to the 5'-untranslated region of the
argA gene located 245 bp upstream of the start codon, and moreover this sequence includes
BamHI recognition site. Primer P33 contains 24 nucleotides homologous to the 5'-region
of the
argA gene located 20 bp upstream of the start codon and start codon itself. The temperature
profile was the following: initial DNA denaturation for 5 min at 95 °C, followed by
25 cycles of denaturation at 95 °C for 30 sec, annealing at 54 °C for 30 sec, elongation
at 72 °C for 1 min 20 sec (for
ArgA gene) or 20 sec(for P
argA promoter) and the final elongation for 5 min at 72 °C. Then, the amplified DNA fragments
was purified by agarose gel-electrophoresis, extracted using "GenElute Spin Columns"
("Sigma", USA) and precipitated by ethanol.
[0178] P
argAArgA fragment was obtained by PCR using both the above-described DNA fragments: P
argA promoter and
ArgA gene. First, the reaction mixture (total volume - 100 µl) consisted of 10 µl of 10x
PCR-buffer with 25 mM MgCl
2 (Sigma, USA), 200 µM each of dNTP and 1 U of Accu-Taq DNA polymerase (Sigma, USA).
The
argA fragment (25 ng) and P
argA (5 ng) were used as a template DNA and as primers simultaneously. Next, primers P31
and P32 were added in reaction mixture. The temperature profile was the following:
1 st step - initial DNA denaturation for 5 min at 95 °C, followed by 10 cycles of
denaturation at 95 °C for 30 sec, annealing at 53 °C for 30 sec, elongation at 72
°C for 1 min, 2nd step - 15 cycles of denaturation at 95 °C, annealing at 54 °C for
30 sec, elongation at 72 °C for 1 min 30 sec. The amplified DNA fragments was purified
by agarose gel-electrophoresis, extracted using "GenElute Spin Columns" ("Sigma",
USA), precipitated by ethanol, treated with
BamHI and
HindIII and ligated with pMW 119/
BamHI- HindIII vector. As a result the plasmid pMW 119-ArgA4 was obtained.
Example 14. The effect of increasing adhE gene expression on L-arginine production
[0179] To evaluate the effect of enhancing expression of the mutant
adhE gene on L-arginine production,
E. coli strains MG1655ΔargR P
L-tacadhE* and MG1655ΔargR were each transformed by plasmid pMW 119- ArgA4. 10 obtained
colonies of each sort of transformants were cultivated at 37°C for 18hours in a nutrient
broth supplemented with 150 mg/l of Ap and 0.1 ml of each of the obtained cultures
was inoculated into 2 ml of fermentation medium in a 20x200 mm test tube and cultivated
at 32°C for 96 hours with a rotary shaker. After cultivation, the amount of L-arginine
which accumulates in the medium was determined by paper chromatography using the following
mobile phase: butanol : acetic acid : water = 4 : 1 : 1 (v/v). A solution (2%) of
ninhydrin in acetone was used as a visualizing reagent. A spot containing L-arginine
was cut out, L-arginine was eluted in 0.5 % water solution of CdCl
2, and the amount of L-arginine was estimated spectrophotometrically at 540 nm. The
results of ten independent test tube fermentations are shown in Table 4. As follows
from Table 4, MG1655ΔargR P
L- tacadhE* produced a higher amount of L-arginine, as compared with MG1655ΔargR P
L-tacadhE*, both in medium with supplemented glucose and without it.
[0180] The composition of the fermentation medium was as follows (g/l):
Ethanol |
20 |
Glucose |
0 / 5 |
(NH4)2SO4 |
25 |
K2HPO4 |
2 |
MgSO4·7H2O |
1.0 |
Thiamine hydrochloride |
0.002 |
Yeast extract |
5.0 |
CaCO3 |
33 |
MgSO4·7H2O, ethanol and CaCO3 were each sterilized separately. |
Table 4
Strain |
Ethanol (2%) |
Ethanol (2%) and glucose (5%) |
OD550 |
Amount of L-arginine, g/l |
OD550 |
Amount of L-arginine, g/l |
MG1655ΔargR(pMW-argAm4) |
1.3±0,2 |
<0.1 |
8.0±0.4 |
1.5±0.2 |
MG 1655ΔargRcat-PL-tac-adhE* (pMW-argAm4) |
7.2±0.4 |
0.8±0.3 |
13.4±0.3 |
1.9±0.2 |
Example 15. Construction of the L-leucine producing E. coli strain NS1391
[0181] The strain NS1391 was obtained as follows.
[0182] At first, a strain having inactivated acetolactate synthase genes (combination
of AilvIH and
ΔilvGM deletions) was constructed. The
ilvIH genes (ΔilvIH::cat) were deleted from the wild-type strain
E. coli K12 (VKPM B-7) by P1 transduction (
Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring
Harbor Laboratory Press (1989).
E. coli MG1655 ΔilvIH::cat was used as a donor strain. Deletion of the
ilvIH operon in the strain MG1655 was conducted by means of the Red-driven integration.
According to this procedure, the PCR primers P34 (SEQ ID NO: 39) and P35 (SEQ ID NO:
40) homologous to the both region adjacent to the
ilvIH operon and gene conferring chloramphenicol resistance in the template plasmid were
constructed. The plasmid pMW-attL-Cm-attR (
PCT application WO 05/010175) was used as a template in a PCR reaction. Conditions for PCR were following: denaturation
step for 3 min at 95 °C; profile for two first cycles: 1 min at 95 °C, 30 sec at 34
°C, 40 sec at 72 °C; profile for the last 30 cycles: 30 sec at 95 °C, 30 sec at 50
°C, 40 sec at 72 °C; final step: 5 min at 72 °C. Obtained 1713 bp PCR product was
purified in agarose gel and used for electroporation of
E. coli MG1655/pKD46. Chloramphenicol resistant recombinants were selected after electroporation
and verified by means of PCR with locus-specific primers P36 (SEQ ID NO: 41) and P37
(SEQ ID NO: 42). Conditions for PCR verification were the following: denaturation
step for 3 min at 94 °C; profile for the 30 cycles: 30 sec at 94 °C, 30 sec at 53
°C, 1 min 20 sec at 72 °C; final step: 7 min at 72 °C. PCR product, obtained in the
reaction with the chromosomal DNA from parental IlvIH
+ strain MG1655 as a template, was 2491 nt in length. PCR product, obtained in the
reaction with the chromosomal DNA from mutant MG1655 ΔilvIH::cat strain as a template,
was 1823 nt in length. As a result the strain MG1655 ΔilvIH::cat was obtained. After
deletion of
ilvIH genes (ΔilvIH::cat) from
E. coli K12 (VKPM B-7) by P1 transduction, Cm
R transductants were selected. As a result the strain B-7 ΔilvIH::cat was obtained.
To eliminate the chloramphenicol resistance marker from B-7 ΔilvIH::cat, cells were
transformed with the plasmid pMW118-int-xis (Ap
R) (
W02005/010175). Ap
R clones were grown on LB agar plates containing 150 mg/l ampicillin at 30°C. Several
tens of Ap
R clones were picked up and tested for chloramphenicol sensitivity. The plasmid pMW118-int-xis
was eliminated from Cm
S cells by incubation on LB agar plates at 42 °C. As a result, the strain B-7 ΔilvIH
was obtained.
[0183] The
ilvGM genes (ΔilvGM::cat) were deleted from
E .
coli B-7 ΔilvIH by P1 transduction. E.
coli MG1655 ΔilvGM::cat was used as a donor strain. The
ilvGM operon was deleted from the strain MG1655 by Red-driven integration. According to
this procedure, the PCR primers P38 (SEQ ID NO: 43) and P39 (SEQ ID NO: 44) homologous
to both the region adjacent to the
ilvGM operon and the gene conferring chloramphenicol resistance in the template plasmid
were constructed. The plasmid pMW-attL-Cm-attR (
PCT application WO 05/010175) was used as a template in the PCR reaction. Conditions for PCR were the following:
denaturation step for 3 min at 95 °C; profile for two first cycles: 1 min at 95 °C,
30 sec at 34 °C, 40 sec at 72 °C; profile for the last 30 cycles: 30 sec at 95 °C,
30 sec at 50 °C, 40 sec at 72 °C; final step: 5 min at 72 °C.
[0184] The obtained 1713 bp PCR product was purified in agarose gel and used for electroporation
of E.
coli MG1655/pKD46. Chloramphenicol resistant recombinants were selected after electroporation
and verified by means of PCR with locus-specific primers P40 (SEQ ID NO: 45) and P41
(SEQ ID NO: 46). Conditions for PCR verification were the following: denaturation
step for 3 min at 94 °C; profile for the 30 cycles: 30 sec at 94 °C, 30 sec at 54
°C, 1 min 30 sec at 72 °C; final step: 7 min at 72 °C. PCR product, obtained in the
reaction with the chromosomal DNA from the parental strain MG1655 as a template, was
2209 nt in length. The PCR product, obtained in the reaction with the chromosomal
DNA from mutant MG1655 ΔilvGm::cat strain as a template, was 1941 nt in length. As
a result, the strain MG1655 ΔilvGm::cat was obtained. After deletion of
ilvGM genes (ΔilvGm::cat) from
E.coli B-7 ΔilvIH by P1 transduction, CmR transductants were selected. As a result the strain
B-7 ΔilvIH ΔilvBN ΔilvGM::cat was obtained. The chloramphenicol resistance marker
was eliminated from B-7 ΔilvIH ΔilvBN ΔilvGM::cat as described above. As a result,
the strain B-7 ΔilvIH ΔilvGM was obtained.
[0185] The native regulator region of the
ilvBN operon was replaced with the phage lambda P
L promoter by the Red-driven integration. For that purpose, the strain B7 ΔilvIH ΔilvGM
with the sole AHAS I was used as an initial strain for such modification. According
to the procedure of Red-driven integration, the PCR primers P42(SEQ ID NO: 47) and
P43 (SEQ ID NO:48) were constructed. Oligonucleotide P42 (SEQ ID NO: 47) was homologous
to the region upstream of the
ilvB gene and the region adjacent to the gene conferring antibiotic resistance which was
present in the chromosomal DNA of BW25113 cat-P
L-yddG. Oligonucleotide P43 (SEQ ID NO: 48) was homologous to both the
ilvB region and the region downstream from the P
L promoter which was present in the chromosome of BW25113 cat-P
L-yddG. Obtaining BW25113 cat-P
L-yddG has been described in detail previously (
EP1449918A1, Russian patent
RU2222596). The chromosomal DNA of strain BW25113 cat-P
L-yddG was used as a template for PCR. Conditions for PCR were the following: denaturation
for 3 min at 95 °C; profile for two first cycles: 1 min at 95 °C, 30 sec at 34 °C,
40 sec at 72 °C; profile for the last 30 cycles: 30 sec at 95 °C, 30 sec at 50 °C,
40 sec at 72 °C; final step: 5 min at 72 °C. As a result, the PCR product was obtained
(SEQ ID NO: 49), purified in agarose gel, and used for electroporation of
E. coli B-7 ΔilvIH ΔilvGM, which contains the plasmid pKD46 with temperature sensitive replication.
Electrocompetent cells were prepared as follows:
E. coli strain B-7 ΔilvIH ΔilvGM was grown overnight at 30 °C in LB medium containing ampicillin
(100 mg/l), and the culture was diluted 100 times with 5 ml of SOB medium (
Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring
Harbor Laboratory Press (1989)) with ampicillin and L-arabinose (1 mM). The cells were grown with aeration at 30
°C to an OD
600 of ≈0.6 and then made electrocompetent by concentrating 100-fold and washing three
times with ice-cold deionized H
2O. Electroporation was performed using 70 µl of cells and ≈100 ng of PCR product.
Following electroporation, the cells were incubated with 1 ml of SOC medium (
Sambrook et al, "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring
Harbor Laboratory Press (1989)) at 37 °C for 2.5 h and after that plated onto L-agar and were grown at 37 °C to
select Cm
R recombinants. Then, to eliminate the pKD46 plasmid, 2 passages on L-agar with Cm
at 42 °C were performed and the obtained colonies were tested for sensitivity to ampicillin.
[0186] The obtained strain B7 ΔilvIH ΔilvGM cat-P
L-ilvBN was valine sensitive. New valine resistant spontaneous mutants of AHAS I were
obtained from this strain. Strains which grew better on 1 g/l of valine were characterized.
[0187] Valine resistance mutations which were resistance to isoleucine were obtained, as
well. Variants with a specific activity which was more than that of the wild-type
were obtained. The nucleotide sequence of the mutant operons for mutant ilvBN4 was
determined. It was revealed that IlvBN4 contained one point mutation in IlvN: N17K
Asn-Lys (codon aac was replaced with aag). Obtained strain B7 ΔilvIH ΔilvGM cat-P
L-ilvBN4 was used for the following constructions.
[0188] Then, cat-P
L-ilvBN4 DNA fragment was transferred from
E .coli B7 ΔilvIH ΔilvGM cat-P
L-ilvBN4 into
E .coli MG1655 mini-Mu::scrKYABR (
EP application 1149911) by P1 transduction. As a result the strain ESP214 was obtained. The chloramphenicol
resistance marker was eliminated from the strain ESP214 as described above. As a result,
the strain ESP215 was obtained.
[0189] Then the DNA fragment shown in (SEQ ID NO: 50) was used for electroporation of the
strain ESP215/pKD46 for the purpose of subsequent integration into chromosome. This
DNA fragment contained regions complementary to the 3' region of the gene
b1701 and to the 5' region of the gene
b1703 (these genes are adjacent to the gene
pps), which are necessary for integration into the chromosome. It also contained an excisable
chloramphenicol resistance marker
cat, and a mutant
ilvBN4 operon under the control of the constitutive promoter P
L. Electroporation was performed as described above. Selected Cm
R recombinants contained a deletion of the gene
pps as a result of the integration of
cat-PL-ilvBN4 fragment into the chromosome. Thus the strain ESP216 was obtained. The chloramphenicol
resistance marker was eliminated from the strain ESP216 as described above. As a result,
the strain ESP217 was obtained.
[0190] At the next step, the mutant
leuA gene (Gly479 -> Cys) under the control of the constitutive promoter P
L was introduced into the strain ESP217. The DNA fragment shown in (SEQ ID NO: 51)
was used for electroporation of the strain ESP217/pKD46 for the purpose of subsequent
integration into the chromosome. This DNA fragment contained the 35nt-region, which
is necessary for integration into the chromosome and homologous to the upstream region
of the gene
leuA. It also contained an excisable region complementary to the sequence of chloramphenicol
resistance marker
cat, and the mutant
leuA (Gly479 -> Cys) gene under the control of the constitutive promoter P
L. Electroporation was performed as described above. Selected Cm
R recombinants contained the mutant gene
leuA (Gly479 -> Cys) under the control of the constitutive promoter P
L integrated into the chromosome. Thus, the strain ESP220 was obtained. The chloramphenicol
resistance marker was eliminated from the strain ESP220 as described above. As a result,
the strain ESP221 was obtained.
[0191] Then, the DNA fragment shown in SEQ ID NO: 52 was used for electroporation of the
strain ESP221/pKD46 for the purpose of subsequent integration into the chromosome.
This DNA fragment contained the 35nt-region homologous to the upstream region of the
gene
tyrB, which is necessary for integration into the chromosome. It also contained an excisable
region complementary to the sequence of chloramphenicol resistance marker
cat and the gene
tyrB with a modified regulatory(-35) region. Electroporation was performed as described
above. Selected Cm
R recombinants contained the gene
tyrB with the modified regulatory(-35) region. Thus, the strain NS1390 was obtained. The
chloramphenicol resistance marker was eliminated from the strain NS1390 as described
above. As a result, the strain NS 1391 was obtained. Leucine producing strain NS1391
was used for further work.
Example 16. The effect of increasing the mutant adhE gene expression on L-leucine production
[0193] Both
E. coli strains, NS1391 and NS1391 P
L-tacadhE*, were cultured for 18-24 hours at 37°C on L-agar plates. To obtain a seed culture,
the strains were grown on a rotary shaker (250 rpm) at 32°C for 18 hours in 20x200-mm
test tubes containing 2 ml of L-broth supplemented with 4% sucrose. Then, the fermentation
medium was inoculated with 0.21 ml of seed material (10%). The fermentation was performed
in 2 ml of a minimal fermentation medium in 20x200-mm test tubes. Cells were grown
for 48-72 hours at 32°C with shaking at 250 rpm. The amount of L-leucine was measured
by paper chromatography (liquid phase composition: butanol - acetic acid - water =
4:1:1). The results of ten independent test tube fermentations are shown in Table
5. As follows from Table 5, NS1391 P
L-tacadhE* produced a higher amount of L-leucine, as compared with NS1391, in media containing
different concentrations of ethanol.
[0194] The composition of the fermentation medium (g/l) (pH 7.2) was as follows:
Glucose |
60.0 |
Ethanol |
0/10.0/20.0/30.0 |
(NH4)2SO4 |
25.0 |
K2HPO4 |
2.0 |
MgSO4·7H2O |
1.0 |
Thiamine |
0.01 |
CaCO3 |
25.0 |
Glucose, ethanol and CaCO3 were sterilized separately. |
Table 5
Strain |
Glucose (6%) |
without ethanol |
+1% ethanol |
+2% ethanol |
+3% ethanol |
Leu,g/l |
OD550 |
Leu,g/l |
OD550 |
Leu,g/l |
OD550 |
Leu,g/l |
OD550 |
NS1391 |
5.0±0.1 |
34.2±0.6 |
4.8±0.1 |
31.7±0.4 |
3.9±0.2 |
31.3±0.6 |
4.0±0.1 |
28.0±0.6 |
NS1391 PL- tac-adhE* |
5.1±0.1 |
31.1±0.2 |
5.9±0.1 |
29.3±0.3 |
4.9±0.1 |
27.3±0.6 |
4.6±0.1 |
22.8±0.2 |
Example 17. The effect of the increasing the adhE gene expression on L-phenylalanine production
[0195] To test the effect of enhanced expression of the
adhE gene under the control of a P
L-tac promoter on phenylalanine production, the DNA fragments from the chromosome of the
above-described strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566 (cl.1); MG1655Δtdh, rhtA*, adhE* can be transferred to the phenylalanine-producing
E. coli strain AJ12739 by P1 transduction (Miller, J.H. (1972) Experiments in Molecular Genetics,
Cold Spring Harbor Lab. Press, Plainview, NY). The strain AJ12739 has been deposited
in the Russian National Collection of Industrial Microorganisms (VKPM) (Russia, 117545
Moscow, 1 Dorozhny proezd, 1) on November 6, 2001 under accession number VKPM B-8197
and then converted to a deposit under the Budapest Treaty on August 23, 2002
[0196] The resulting strains and the parent strain AJ12739 can each be cultivated at 37
°C for 18 hours in a nutrient broth, and 0.3 ml of the obtained cultures can each
be inoculated into 3 ml of a fermentation medium in a 20 x 200 mm test tube and cultivated
at 37 °C for 48 hours with a rotary shaker. After cultivation, the amount of phenylalanine
which accumulates in the medium can be determined by TLC. 10 x 15 cm TLC plates coated
with 0.11 mm layers of Sorbfil silica gel without fluorescent indicator (Stock Company
Sorbpolymer, Krasnodar, Russia) can be used. The Sorbfil plates can be developed with
a mobile phase: propan-2-ol : ethylacetate : 25% aqueous ammonia : water = 40 : 40
: 7 : 16 (v/v). A solution (2%) of ninhydrin in acetone can be used as a visualizing
reagent.
[0197] The composition of the fermentation medium (g/l):
Ethanol |
20.0 |
(NH4)2SO4 |
16.0 |
K2HPO4 |
0.1 |
MgSO4·7H2O |
1.0 |
FeSO4·7H2O |
0.01 |
MnSO4·5H2O |
0.01 |
Thiamine HCl |
0.0002 |
Yeast extract |
2.0 |
Tyrosine |
0.125 |
CaCO3 |
20.0 |
[0198] Ethanol and magnesium sulfate are sterilized separately. CaCO
3 dry-heat sterilized at 180 °C for 2 hours. pH is adjusted to 7.0.
Example 18. The effect of increasing the adhE gene expression on L-tryptophan production
[0199] To test the effect of enhanced expression of the
adhE gene under the control of a P
L-tac promoter on tryptophan production, the DNA fragments from the chromosome of the above-described
strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566(cl.1); MG1655Δtdh, rhtA*, adhE* can be transferred to the tryptophan-producing
E. coli strain SV164 (pGH5) by P1 transduction (
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,
Plainview, NY). The strain SV164 has the
trpE allele encoding anthranilate synthase which is not subject to feedback inhibition
by tryptophan. The plasmid pGH5 harbors a mutant
serA gene encoding phosphoglycerate dehydrogenase which is not subject to feedback inhibition
by serine. The strain SV164 (pGH5) is described in detail in
U.S. Patent No. 6,180,373 or European patent
0662143.
[0200] The resulting strains and the parent strain SV 164 (pGH5) can each be cultivated
with shaking at 37 °C for 18 hours in 3 ml of nutrient broth supplemented with 20
mg/l of tetracycline (marker of pGH5 plasmid). 0.3 ml of the obtained cultures can
each be inoculated into 3 ml of a fermentation medium containing tetracycline (20
mg/l) in 20 x 200 mm test tubes, and cultivated at 37 °C for 48 hours with a rotary
shaker at 250 rpm. After cultivation, the amount of tryptophan which accumulates in
the medium can be determined by TLC as described in Example 17. The fermentation medium
components are set forth in Table 6, but should be sterilized in separate groups A,
B, C, D, E, F, and H, as shown, to avoid adverse interactions during sterilization.
Table 6
Solutions |
Component |
Final concentration, g/l |
A |
KH2PO4 |
1.5 |
|
NaCl |
0.5 |
|
(NH4)2SO4 |
1.5 |
|
L-Methionine |
0.05 |
|
L-Phenylalanine |
0.1 |
|
L-Tyrosine |
0.1 |
|
Mameno (total N) |
0.07 |
B |
Ethanol |
20.0 |
|
MgSO4·7H2O |
0.3 |
C |
CaCl2 |
0.011 |
D |
FeSO4·7H2O |
0.075 |
|
Sodium citrate |
1.0 |
E |
Na2MoO4·2H2O |
0.00015 |
|
H3BO3 |
0.0025 |
|
CoCl2·6H2O |
0.00007 |
|
CuSO4·5H2O |
0.00025 |
|
MnCl2·4H2O |
0.0016 |
|
ZnSO4·7H2O |
0.0003 |
F |
Thiamine HCl |
0.005 |
G |
CaCO3 |
30.0 |
H |
Pyridoxine |
0.03 |
Solution A had a pH of 7.1, adjusted by NH4OH. |
Example 19. The effect of the increasing the adhE gene expression on L-histidine production
[0201] To test the effect of enhanced expression of the
adhE gene under the control of a P
L-tac promoter on histidine production, the DNA fragments from the chromosome of the above-described
strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566(cl.1); MG1655Δtdh, rhtA*, adhE* can be transferred to the histidine-producing
E. coli strain 80 by P1 transduction (
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,
Plainview, NY). The strain 80 has been described in Russian patent
2119536 and deposited in the Russian National Collection of Industrial Microorganisms (Russia,
117545 Moscow, 1 Dorozhny proezd, 1) on October 15, 1999 under accession number VKPM
B-7270 and then converted to a deposit under the Budapest Treaty on July 12, 2004.
[0202] The resulting strains and the parent strain 80 can each be cultivated in L broth
for 6 hours at 29 °C. Then, 0.1 ml of obtained culture can each be inoculated into
2 ml of fermentation medium in a 20x200mm test tube and cultivated for 65 hours at
29 °C with a rotary shaker (350 rpm). After cultivation, the amount of histidine which
accumulates in the medium can be determined by paper chromatography. The paper can
be developed with a mobile phase: n-butanol : acetic acid : water = 4 : 1 : 1 (v/v).
A solution of ninhydrin (0.5%) in acetone can be used as a visualizing reagent.
[0203] The composition of the fermentation medium (pH 6.0) (g/l):
Ethanol |
20.0 |
Mameno (soybean hydrolyzate) |
0.2 as total nitrogen |
L-proline |
1.0 |
(NH4)2SO4 |
25.0 |
KH2PO4 |
2.0 |
MgSO4·7H2O |
1.0 |
FeSO4·7H2O |
0.01 |
MnSO4 |
0.01 |
Thiamine |
0.001 |
Betaine |
2.0 |
CaCO3 |
60.0 |
Ethanol, proline, betaine and CaCO3 are sterilized separately. pH is adjusted to 6.0 before sterilization. |
Example 20. The effect of increasing the adhE gene expression on L-glutamic acid production
[0204] To test the effect of enhanced expression of the
adhE gene under the control of a P
L-tac promoter on glutamic acid production, the DNA fragments from the chromosome of the
above-described strains MG1655Δtdh, rhtA*, P
L-tacadhE; MG1655Δtdh, rhtA*, P
L-tacadhE*; MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568 (cl.18); MG1655Δtdh, rhtA*, P
L-tacadhE-Lys568, Val566 (cl.1); MG1655Δtdh, rhtA*, adhE* can be transferred to the glutamic
acid-producing
E. coli strain VL334thrC
+ (
EP1172433) by P1 transduction (
Miller, J.H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Lab. Press,
Plainview, NY). The strain VL334thrC
+ has been deposited in the Russian National Collection of Industrial Microorganisms
(VKPM) (Russia, 117545 Moscow, 1 Dorozhny proezd, 1) on December 6, 2004 under the
accession number VKPM B-8961 and then converted to a deposit under the Budapest Treaty
on December 8, 2004.
[0205] The resulting strains and the parent strain VL334thrC
+ can each be cultivated with shaking at 37 °C for 18 hours in 3 ml of nutrient broth.
0.3 ml of the obtained cultures can each be inoculated into 3 ml of a fermentation
medium in 20 x 200 mm test tubes, and cultivated at 37 °C for 48 hours with a rotary
shaker at 250 rpm.
[0206] The composition of the fermentation medium (pH 7.2) (g/l):
Ethanol |
20.0 |
Ammonium sulfate |
25.0 |
KH2PO4 |
2.0 |
MgSO4·7H2O |
1.0 |
Thiamine |
0.0001 |
L-isoleucine |
0.05 |
CaCO3 |
25.0 |
Ethanol and CaCO3 were sterilized separately. |
Industrial Applicability
[0207] According to the present invention, production of an L-amino acid by a bacterium
of the
Enterobacteriaceae family can be enhanced.
SEQUENCE LISTING
[0208]
<110> Ajinomoto Co., Inc.
<120> A METHOD FOR PRODUCING AN L-AMINO ACID USING A BACTERIUM OF THE ENTEROBACTERIACEAE
FAMILY
<130> C666-C7177
<150> RU2006125964
<151> 2006-07-19
<150> US60/885671
<151> 2007-01-19
<160> 58
<170> Patent In version 3.1
<210> 1
<211> 2676
<212> DNA
<213> Escherichia coli
<220>
<221> CDS
<222> (1)..(2676)
<400> 1
<210> 2
<211> 891
<212> PRT
<213> Escherichia coli
<400> 2
<210> 3
<211> 70
<212> DNA
<213> Artificial
<220>
<223> primer P1
<400> 3
<210> 4
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P2
<400> 4
<210> 5
<211> 18
<212> DNA
<213> Artificial
<220>
<223> primer P3
<400> 5
cggtcatgct tggtgatg 18
<210> 6
<211> 21
<212> DNA
<213> Artificial
<220>
<223> primer P4
<400> 6
ttaatcccag ctcagaataa c 21
<210> 7
<211> 183
<212> DNA
<213> Artificial
<220>
<223> hybrid promoter
<400> 7
<210> 8
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P5
<400> 8
<210> 9
<211> 58
<212> DNA
<213> Artificial
<220>
<223> primer P6
<400> 9
attagtaaca gccataatgc tctcctgata atgttaaacc gctcacaatt ccacacat 58
<210> 10
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer P7
<400> 10
acttgttctt gagtgaaact ggca 24
<210> 11
<211> 22
<212> DNA
<213> Artificial
<220>
<223> primer P8
<400> 11
aagacgcgct gacaatacgc ct 22
<210> 12
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P9
<400> 12
<210> 13
<211> 65
<212> DNA
<213> Artificial
<220>
<223> primer P10
<400> 13
<210> 14
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer P11
<400> 14
aagacgcgct gacaatacgc cttt 24
<210> 15
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer P12
<400> 15
aaggggccgt ttatgttgcc agac 24
<210> 16
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P13
<400> 16
<210> 17
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P14
<400> 17
<210> 18
<211> 67
<212> DNA
<213> Artificial
<220>
<223> primer P15
<400> 18
<210> 19
<211> 23
<212> DNA
<213> Artificial
<220>
<223> primer P16
<400> 19
cttcgaagta gaagcggacc cga 23
<210> 20
<211> 27
<212> DNA
<213> Artificial
<220>
<223> primer P17
<400> 20
ccagaagtgg tggtgacagc gatcatt 27
<210> 21
<211> 75
<212> DNA
<213> Artificial
<220>
<223> primer P18
<400> 21
<210> 22
<211> 73
<212> DNA
<213> Artificial
<220>
<223> primer P19
<400> 22
<210> 23
<211> 31
<212> DNA
<213> Artificial
<220>
<223> primer P20
<400> 23
atcgaattca agacgcgctg acaatacgcc t 31
<210> 24
<211> 27
<212> DNA
<213> Artificial
<220>
<223> primer P21
<400> 24
cacgctctac gagtgcgtta agttcag 27
<210> 25
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P22
<400> 25
<210> 26
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer P23
<400> 26
ttcgaattcg ttgtgtctca aaatctccga 30
<210> 27
<211> 21
<212> DNA
<213> Artificial
<220>
<223> primer P24
<400> 27
cgtcttcaga cagaacacca c 21
<210> 28
<211> 23
<212> DNA
<213> Artificial
<220>
<223> primer P25
<400> 28
atgcttgatg gtcggaagag gca 23
<210> 29
<211> 2688
<212> DNA
<213> Pantoea ananatis
<220>
<221> CDS
<222> (1)..(2688)
<400> 29
<210> 30
<211> 895
<212> PRT
<213> Pantoea ananatis
<400> 30
<210> 31
<211> 69
<212> DNA
<213> Artificial
<220>
<223> primer P26
<400> 31
<210> 32
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P27
<400> 32
<210> 33
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer P28
<400> 33
aatcccgctc tttcataaca ttat 24
<210> 34
<211> 23
<212> DNA
<213> Artificial
<220>
<223> primer P29
<400> 34
attaatcgca ggggaaagca ggg 23
<210> 35
<211> 42
<212> DNA
<213> Artificial
<220>
<223> primer P30
<400> 35
atcatgcaaa gaggtgtgcc gtggtaaagg aacgtaaaac cg 42
<210> 36
<211> 42
<212> DNA
<213> Artificial
<220>
<223> primer P31
<400> 36
atcatgcaaa gaggtgtgcc gtggtaaagg aacgtaaaac cg 42
<210> 37
<211> 30
<212> DNA
<213> Artificial
<220>
<223> primer P32
<400> 37
gttggatcct gacatgcctc tcccgagcaa 30
<210> 38
<211> 24
<212> DNA
<213> Artificial
<220>
<223> primer P33
<400> 38
ccacggcaca cctctttgca tgat 24
<210> 39
<211> 61
<212> DNA
<213> Artificial
<220>
<223> primer P34
<400> 39
<210> 40
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P35
<400> 40
<210> 41
<211> 21
<212> DNA
<213> Artificial
<220>
<223> primer P36
<400> 41
ttgctgtaag ttgtgggatt c 21
<210> 42
<211> 20
<212> DNA
<213> Artificial
<220>
<223> primer P37
<400> 42
tccaggttcc cactgatttc 20
<210> 43
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P38
<400> 43
<210> 44
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P39
<400> 44
<210> 45
<211> 20
<212> DNA
<213> Artificial
<220>
<223> primer P40
<400> 45
tggtcgtgat tagcgtggtg 20
<210> 46
<211> 20
<212> DNA
<213> Artificial
<220>
<223> primer P41
<400> 46
cacatgcacc ttcgcgtctt 20
<210> 47
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P42
<400> 47
<210> 48
<211> 64
<212> DNA
<213> Artificial
<220>
<223> primer P43
<400> 48
<210> 49
<211> 1968
<212> DNA
<213> artificial
<220>
<223> DNA fragment containing cat gene and PL promoter
<400> 49
<210> 50
<211> 4971
<212> DNA
<213> DNA fragment
<400> 50
<210> 51
<211> 3786
<212> DNA
<213> DNA fragment
<400> 51
<210> 52
<211> 2942
<212> DNA
<213> DNA fragment
<400> 52
<210> 53
<211> 891
<212> PRT
<213> Shigella flexneri
<400> 53
<210> 54
<211> 891
<212> PRT
<213> Yersinia pestis
<400> 54
<210> 55
<211> 891
<212> PRT
<213> Erwinia carotovora
<400> 55
<210> 56
<211> 878
<212> PRT
<213> Salmonella typhimurium
<400> 56
<210> 57
<211> 867
<212> PRT
<213> Lactobacillus plantarum
<400> 57
<210> 58
<211> 903
<212> PRT
<213> Lactococcus lactis
<400> 58